Preclinical Drug Discovery Blog

Can a preclinical model of psoriasis translate to human psoriasis?

MD Biosciences — Mon, 29 Apr 2013 21:28:00 GMT

First let's start by talking about what makes a good preclinical model of psoriasis?

A good pre-clinical psoriasis model should obviously re-capitulate the key features of the clinical disease in humans. Therefore, a plausible model will exhibit the following criteria:

Epidermal hyperproliferation of keratinocytes (acanthosis) and altered differentiation of the epidermis.
Papillomatosis (regular and symmetrical extension of rete ridges).
Infiltration of T cells, dendritic cells, macrophages and neutrophils.
Functional role for T cells.
Altered dermal vascularity.

From a commercial viewpoint, other criteria can be added to the list such as the chosen model should also be rapid, convenient and cost effective for screening psoriatic drugs. To date, this has been an aspect that researchers have struggled with and many utilize complex and costly xenograft and trasngenic models.

Imiquimod-induced preclinical model of psoriasis-like inflammation

Topical treatment of skin with Aldara, a cream preparation containing 5% imiquimod (IMQ), results in tumor regression in up to 90% of patients with non-melanoma skin cancer. IMQ is a ligand for the toll-like receptors TLR7 and TLR8. It is a potent immune activator that is commonly used for virus-associated skin abnormalities and cancerous lesions. It exacerbates psoriasis at both the local treated areas as well as distant sites, which has led to the development of a pre-clinical model of psoriasis using topically applied Aldara cream. The anti-tumor and anti-viral effects of IMQ are mostly mediated by activation of TLR7 and TLR8 expressed by monocytes, macrophages and dendritic cells producing pro-inflammatory cytokines and chemokines. Application of Aldara on the ears and back of mice results in the development of psoriasis-like lesions within 5 days of application and is underpinned by an influx of various cells as well as hyperplasia of the epidermis.

How does the IMQ-induced psoriasis-like inflammation model correlate to human psoriasis?

Although it is recognized that mouse skin differs from human skin in several ways, a recent study employing functional genomics methods has revealed many similaries between human psoriais and mouse models across thousands of genes thus supporting the use of preclinical models to screen anti-psoriatic compounds. Over 50,000 transcripts represented on the Affymetrix Human genome were compared to corresponding orthologues in the Affymetric Mouse genome. This comparative study demonstrated that there was high correlation between psoriatic gene expression in human psoriatic skin samples in comparison to the IMQ-induced psoriasis model. In particular, these similarities were related to genes responsible for epidermal development and keratinization.

Benefits of the IMQ-induced psoriasis model compared to other psoriasis models?

The IMQ-induced psoriasis model represents a simply, rapid and cost effective method of inducing psoriasis in mice, which avoids the expense and labor intensive breeding programs, which are required for producing the keratinocyte transgenic mouse lines (e.g. K14-AREG, K5-Stat3C or K5-TGFb1) or the complexity and expertise required for the xenograft models. Furthermore, some of these transgenic mouse lines suffer from shortened life span due to stunted growth and severe psoriatic skin lesions as a result of the genetic modification, thus rendering them less amenable to therapeutic intervention.

Download the IMQ-induced Psoriasis Whitepaper:

Background of psoriasis pathology
Models of pre-clinical psoriasis and correlation to human
Data on psoriasis score, histology, spleen weights and cell composition, biomarkers.

Could an In vitro herpes infection model incorporate HSV latency?

MD Biosciences — Tue, 23 Apr 2013 15:39:00 GMT

HSV Background
Herpes simplex virus (HSV) infection is among the most common skin disorders with more than 90% of the population infected with HSV. The most common manifestation is cold sores although it can cause diseases of other organs which are more serious. Once the initial acute infection is treated, the virus will persist in the human body in a latent form that can be reactivated upon various factors such as UV exposure or stress. The anti-viral therapies to date tend to alleviate the symptoms and shorten the infection but do little to prevent the reactivation of the virus.

The challenge with HSV latency

After the initial infection, the HSV will migrate into the cell body of the sensory neurons that innervate the infected region. HSV enters the ganglia over the axons of the nerve cells and becomes latent in the ganglion. During latency, there is no virus replication and the viral DNA persists undetected by the immune system of the host. The challenge of current in vitro HSV systems is that they are mainly indicative of the intial infection and don't reflect the dormancy or reactivation of the virus. Many studies therefore default to in vivo models. We set out to evaluate the ability to have an in vitro system that reflects the latency of the HSV-1 virus.

In vitro HSV assay that reflects the human infection

The basis of the assay is to incorporate the latent virus in the skin equivalent model to enable the evaluation of anti-viral therapies. The platform of the assay is run on the 3D skin equivalent system which is a full skin equivalent (FSE) containing a dermis and epidermal layer. The FSE is co-cultured with a neuronal PC-12 cell line that has been infected with HSV-1. Viral DNA and cell latency is verified with PCR and TCID50 assay. Once the infected PC-12 neuronal cell line has been integrated into the collagen matrix dermal layer of the FSE, the virus can be reactivated by a UV light. Compounds can then be applied to test the anti-viral efficacy against the reactivated HSV.

Figure 3A shows the successful integration of latently infected PC12 cells within the 3D skin equivalent with HSV-1 neuronal cells.

Figure 3B shows the detection of neuronal PC12 cells was performed using a specific antibody (Fig. 3B).

Figure 3C shows isotype control

Further developing the 3D Full Skin Equivalent HSV-1 model

MD Biosciences in conjunction with the Fraunhofer institute is able to offer an in vitro model that reflects the dormancy of HSV-1. Further validation is required to assess the reproducibility of the reactivation and efficacy of anti-viral agents. If you would like to know more about the model or consider joining in the validation studies, please contact us.

References:

Lycke, E.; Hamark, B.; Johansson, M.; Krotochwil, A.; Lycke, J.; Svennerholm, B. (1988) Herpes simplex virus infection of the human sensory neuron. An electron microscopy study. Arch Virol. 101(1-2): 87-104
Topp, K. S.; Meade, L. B.; LaVail, J. H. (1994) Microtubule polarity in the peripheral processes of trigeminal ganglion cells: relevance for the retrograde transport of herpes simplex virus. J Neurosci. 14(1): 318-325
Decman, V.; Freeman, M. L., Kinchington, P. R.; Hendricks,R. L. (2005) Immune control of HSV-1 latency. Viral Immunol. 18(3): 466-473

The 3D Skin Model is developed and patented by Fraunhofer IGB. Commercialization is by MD Biosciences, Inc.

Contributions of the preclinical 6OHDA model to understanding PD

MD Biosciences — Mon, 04 Mar 2013 16:23:00 GMT

Background of the preclinical 6-OHDA model of PD

The 6-hydroxydopamine (6-OHDA) model of Parkinson's Disease (PD) was the first model of PD generated, and has since been widely used to investigate parkinsonism in rodents. The model was originally developed following the discovery that injecting 6-OHDA into the substantia nigra pars compacta (SNpc) caused anterograde degeneration of the nigrostriatal dopaminergic system, producing a loss of dopaminergic neurons in the SNpc and loss of dopaminergic terminals in the striatum (to which the SNpc projects), similar to that observed in Parkinson’s disease. 6-OHDA is similar in structure to dopamine, but the presence of an additional hydroxyl group makes it toxic to dopaminergic neurons. Once in the cytosol, 6-OHDA auto-oxidizes to form reactive oxygen species, which are thought to cause neurodegeneration by reducing levels of anti-oxidant enzymes, elevating iron, and inhibiting mitochondrial respiration. The main features of this model that have made it popular are that it is relatively fast, inexpensive and simple to implement, and that the lesions it produces are reproducible and substantial.

Contributions to understanding and treating PD

The 6-OHDA model has contributed significantly to our understanding of Parkinson's Disease (PD) and the development of treatments. It has been used to help understand the mechanisms of PD pathogenesis and the roles that neurodegenerative processes, such as free radical generation, energy crisis, complex I blockade, and inflammation have in PD. The model has been used to test the efficacy of new experimental therapeutics, including those designed to treat motor symptoms, slow cell death, replace lost neurons and alleviate dyskinetic side effects. It has helped improve understanding of the physiology and circuitry of the basal ganglia, the role of dopamine receptor subtypes and the side effects of various PD treatments. And, it has contributed to the development of L-dopa therapy and deep brain stimulation. Many of the drugs in clinical use today, with the exception of anticholinergic medications, have shown efficacy in 6-OHDA lesion models [1].

Although the model also has been employed to assess neuroprotective strategies, its usefulness in this regard has not been demonstrated because there is no neuroprotective strategy for PD.

Assessing validity of the 6-OHDA model

In vitro models attempt to capture as many of the hallmarks of PD as possible. But because no model can recapitulate all the behavioral, histopathological or molecular aspects of a disease, models must be selected on the basis of being the most suitable for the question being asked and the specific objectives being pursued.

A key measure of the performance of any model is model validity, assessed from the three perspectives of face validity, construct validity and predictive validity. The 6-OHDA model scores well in all three categories.

Face validity assesses how well the model reproduces the symptomatic and histopathological hallmarks of the human disease, such as rigidity, tremor, postural instability, bradykinesia, akinesia, as well as selective degeneration of dopaminergic neurons, dopamine depletion, and the presence of Lewy bodies. Ideally the model would also mirror the more minor characteristics of the disease, such as the lesion of non-dopaminergic neurons, involvement of other transmitter systems, and the non-motor symptoms like as cognitive decline, depression and anxiety.

The 6-OHDA rat model reproduces well the neurodegenerative profile and dopamine-related features of the disorder, but the presence of Lewy bodies, the pathological hallmark of PD, has never been observed. The rat behavioral and cognitive repertoire will never mirror that of the human, but some PD symptoms, e.g. akinesia, bradykinesia and postural instability lend themselves to examination by the 6-OHDA model. It is still not clear whether the 6-OHDA model shows face validity for the non-motor symptoms.

Construct validity examines similarities in pathogenesis between the model and the disorder, e.g. in the case of PD, the involvement of oxidative stress, respiratory inhibition, and inflammation. In PD patients increased oxidative stress, diminished levels of anti-oxidant enzymes, mitochondrial dysfunction (i.e. respiratory inhibition), an increase in inflammatory markers, and activated microglia have all been observed in dopaminergic neurons.

Although the mechanism of 6-OHDA toxicity is not completely understood, it is known that oxidative stress, mitochondrial dysfunction and probably inflammation are involved. In the cytosol, 6-OHDA oxidizes to form reactive oxygen species that reduce the levels of antioxidant enzymes, raise iron levels, and interact with complexes I and IV of the mitochondrial respiratory chain, leading to further oxidative stress. Inflammatory processes have been suggested by studies using the PET ligand PK11195, which showed activated microglia in the striatum and SN of 6-OHDA lesioned rats [reviewed in 1-3].

Predictive validity asks whether the model can identify agents that will be clinically effective. The 6-OHDA model has shown predictive validity for many anti-parkinsonian drugs in use today with the exception of the anticholinergics [1, table 1]. Good predictive validity for neuroprotective strategies has not been demonstrated because there is no neuroprotective strategy for PD.

Over the next few weeks, we will continue discussing the 6-OHDA model, it benefits, drawbacks, various sites of dosing and outcomes expected as well as the behavior tests and what they tell us.

What animal models of Parkinson's Disease have revealed about pathogenesis and treatment. Click here to download free whitepaper.

References:

Duty, S. & Jenner, P. Animal models of Parkinson’s disease: a source of novel treatments and clues to the cause of the disease. British journal of pharmacology 164, 1357–91 (2011).
Bové, J. & Perier, C. Neurotoxin-based models of Parkinson’s disease. Neuroscience 211, 51–76 (2012).
Schober, A. Classic toxin-induced animal models of Parkinson’s disease: 6-OHDA and MPTP. Cell and tissue research 318, 215–24 (2004).

Bioimaging in preclinical models of Rheumatoid Arthritis

MD Biosciences — Mon, 05 Nov 2012 17:12:00 GMT

Rheumatoid arthritis is a chronic and progressive inflammatory condition estimated to affect between 0.5-1% of the world's population, with more women being affected than men. It is a systemic disease manifesting mainly as a disabiling destruction of the synovial joints of the hands and feet.

Joint destruction is induced by dysregulated immune activation of both the innate and adaptive immune responses resulting in alterations in the synovium, cartilage and bone. The normal joint has a thin synovial lining (intimal lining layer), 1-3 cells thick. Beneath this is a sub-lining layer of connective tissue scattered with immune cells, blood vessels and nerve cells. Together these layers form the synovium, which produces the synovial fluid that serves to lubricate the joint. Disease initiation results in profound changes in the structure and composition of the synovium and synovial fluid; with the infiltration of inflammatory cells, synovial cell hyperplasia, increased angiogenesis, fibroblast proliferation and extracellular matrix production. This increase in synovial cell proliferation can result in the lining increasing up to five times its original size and can result in pannus formation. The culmination of these events is bone and cartilage erosion and loss of joint function.

Research spanning five decades as failed to completely elucidate the aetiology of RA. It is clear however that the disease is complex, heterogeneous and can probably be initiated by several mechanisms. The strongest is with HLA II, although both genetic and environmental factors have been implicated in the disease.

Many preclinical models of rheumatoid arthritis are used to study the mechanisms of the disease and to screen potentially therapeutic targets.

Models Inducing Rheumatoid Arthritis	Models of Spontaneous Arthritis
Collagen-induced Arthritis (CIA)	TNFa overexpressing transgenic (Tg) mice
Collagen Antibod-induced Arthritis (CAIA)	K/BxN mice
Adjuvant-induced Arthritis (AIA)	SKG mice
Zymosan-induced Arthitis	Human DR4-CD4 mice
Streptococcal cell wall-induced Arthritis	IL-1Ra -/- mice

While these models have greatly advanced our understanding and treatment of inflammatory conditions such as RA, pharma companies continue to face enormous challenges bringing new drugs to market. It is estimated to cost $0.8-$1.7 billion dollars for a new drug to reach the market, however 4 out of 5 drugs that enter clinical trials fail to proceed to approval stages, representing a substantial financial loss to the company. Advances in imaging technology has however, has allowed these models to provide significantly better information about disease and potential disease modifying therapies (DMARD). This can be used to help decide which drugs should advance to clinical stages by providing accurate information about drug localization, mechanisms of action, receptor occupancy, effect on specific molecules, process and pathways and efficacy of dose and regime.

Using inflammation specific probes in the collagen-induced arthritis (CIA) pre-clinical model, researchers can:

perform repeated measures of arthritis associated inflammation markers temporally in the same animal without the need to add extra animals
Multiple joints can be observed including the knee which can be difficult to visualize using standard arthritis clinical scoring protocols
Highlight disease processes earlier in the course of arthritis development
Make decisions during the course of the study based on biomarker imaging that may not be detected by clinical scoring

Figure: Cathepsin B, L, S, K and plasmin detected with Bioimaging Inflammation probes

Post-stroke neuroinflammation: astrocytes & microglia

MD Biosciences — Mon, 09 Jul 2012 17:22:00 GMT

Post-stroke neuroinflammation is a very complex phenomenon involving multiple resident and invading cell types at varying degrees of differentiation or activation - each expressing specific subsets of diffusable factors, receptors, cellular adhesion molecules and other markers - all of which is changing as time passes to create an initially neurotoxic and then finally neuroprotective environment. This inflammatory process in the penumbra offers a broad array of potential cellular and molecular targets with much wider therapeutic windows.

At the cellular level, neurons, microglia, astrocytes and cerebrovascular endothelial cells are the first affected by the ischemic conditions and their responses to massive cell death in neighboring tissue initiates the precisely timed arrival of successive subsets of leukocytes - first neutrophils, followed by monocytes, macrophages and finally T cels. Targeting these cells via manipulation of their phenotypes, activation states, movements into lesion sites ro their release of harmful mediators represents a major investigative pathway toward potential therapeutics for ischemic stroke sufferers.

We will break down the involvement of the various cells in this post-stroke neuroinflammatory environment, starting with microglia and astrocytes.

Microglia

Microglia are the first to detect and respond to ischemic conditions, showing signs of activation as early as a few minutes after infarct. Microglia are the resident brain immunocompetent cells and constitutively express a wide variety of pattern recognition receptors. After stroke, resting microglia detect the spilled cytoplasmic contents of necrotic cells as well as high levels of reactive oxygen species (ROS) and consequently transform to an active, phagocytic phenotype, functioning to clear dead cells, cellular debris and release pro-inflammatory and cytotoxic diffusible factors. Inhibitors of microglial activation, such as the multi-functional drug Minocycline, have been shown to reduce infarct size in preclinical stroke models when administered intraveneously in combination with rt-PA within 6 hours of infarct. At later timepoints, it appears microglia have neuroprotective effects, such as producing neurotrophic and other growth factors supporting neurogenesis and neural plasticity.

Astrocytes

Astrocytes normally provide energy, neurotrophic and neurogenic factors, neurotransmitter precursors and anti-oxidant defense to neurons. Like microglia, astrocytes also proliferate and differentiate in response to stroke and begin to produce pro-inflammatory and cytotoxic mediators as well as contribute to the loss of the blood-brain barrier (BBB) integrity. Astroglial activity begins at the core approximately 4 hours after stroke and may be observed for up to 28 days. Modulators of astrocyte function such as Arundic Acid appear to improve outcomes in preclinical stroke models. Like microglia, astrocyte activity appears to be initially damaging and later protective.

The complex interplay between resident and invading cells and their bioactive effector signalling molecules as they intially damage and later protect and repair brain tissue offers many opportunities for pharmacological intervention in clinical feasible therapeutic windows. Download the full eBook to read more.

MD Biosciences offers preclinical stroke models for ischemic stroke research. Contact us to discuss a custom preclinical study design.

Candelerio-Jalil, E. (2009) Current Opinion in Investigational Drugs 10(7):644
Ceulemans, A.G., Zgvac, T., Kooijman, R., Hachimi-Idrissi, S., Sarre, S., Michotte, Y (2010) Journal of Neuroinflammation 7:74
Downes, CE. and Crank, P.J. (2010) British Journal of Pharmacology 160:1872
Lakhan, S.E., Kirchgessner, A., and Hofer, M. (2009) The Open Neurology Journal 4:34

MMPs in ischemic conditions (stroke and myocardial infarct)

MD Biosciences — Mon, 25 Jun 2012 19:20:00 GMT

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that function to degrade extracellular matrix. The turnover of ECM in organs is regulated by a balance by MMPs and their inhibitors (TIMPs) and the imbalance is implicated in a variety of diseases. Here we focus on the roles of MMPs in ischemia - particularly cerebral stroke and myocardial infarct.

MMPs in cerebral ischemia

Two MMP family members, MMP-2 and MMP-9, are known to degrade the major components of the BBB basal lamina (i.e. type IV collagen, laminin and fibronectin) and have been implicated in ischemic stroke. Inflammatory cytokines repidly up-regulate MMP-9 first in endothelial cells, then in neutrophils (which degranulate releasing pro- and active forms of MMP-9) and finally in macrophages. MMP-9 knock-out and neutrophil degranulation suppression strategies in preclinical cerebral ischemia models have been shown to reduce stroke damage. Futher, SB-3CT, a selective inhibitor of MMP-2 and MMP-9, appears to reduce lesion volume in preclinical models of stroke. Additionally, statin drugs and Minocycline both appear to have inhibitory effects on MMPs and are known to improve outcomes in preclinical cerebral stroke models. Researchers are focused on MMPs and how they contribute to the pathological events in the brain following stroke, traumatic brain injury and other neurodegenerative effects.

Download our complimentary eBook that covers other inflammatory events following Acute Ischemic stroke

MMPs in myocardial ischemia

Early or exaggerated degradation of existing ECM or late/incorrect generation of new ECM can jeopardize the structural integrity of the heart leading to geometric changes in the LV that make it more susceptible to rupture, dilation, arrhythmias and pump failure. After myocardial infarct (MI), pro-inflammatory cytokines including TNFa and IL-1b act to increase both the expression and the activity of MMPs in cardiac tissues, resulting in proteolytic cleavage of a variety of ECM proteins. In addition to their prominent role in matrix degradation, MMPs also exacerbate the post-MI inflammatory environment in a number of ways. MMPs are responsible for post-translational processing of many cytokines, chemokines and growth factors - such as shedding them from membranes to create soluble forms, activating them by cleaving pro regions or making other modifications, many of which contribute to the promotion of inflammation. Low molecular weight ECM fragments generated by MMP proteolytic activity are detectable in circulation within minutes of myocardial infarct. This ECM debris has pro-inflammatory properties and is capable of eliciting leukocyte chemotaxis with some evidence sugging that collagen fragments may signal through CXCR1 and CXCR2 chemokine receptors on neutrophils and low-molecular with hyaluronan fragments may infuced pro-inflammatory cytokine expression in macrophages and endothelial cells via interactions with CD44. As inflammation resolves, anti-inflammatory cytokines, including TGFb, up-regulate expression of Tissue Inhibitor of Metalloroteinases (TIMPs) allowing newly-generated matrix to be preserved. A significant amount of experimental evidence from preclinical myocardial ischemia models suggests that MMP inhibitors may have cardioprotective effects (which has yet to be observed in the clinical setting).

Download our eBook: Inflammation following acute myocardial infarct to review other inflammatory factors following MI.

Post Myocardial Infarct (MI) inflammatory environment

MD Biosciences — Mon, 11 Jun 2012 19:44:00 GMT

At the tissue level, inflammation ensues very rapidly after myocardial infarct (MI), initially prompted by detection of high levels of reactive oxygen species (ROS) and necrotic cellular debris by resident cells in neighboring non-infarct tissues. ROS and necrotic cell debris are also detected by peripheral leukocytes, which home to the injured tissue, exit circulation, and infiltrate infarct and non-infarct tissues. Upon entering the lesion site, these leukocytes further release ROS, proteolytic enzymes, pro-inflammatory and cytotoxic diffusible factors and participate in phagocytosis of necrotic cells and disrupted ECM. This post-MI inflammatory environment in cardiac tissues peaks at 1 to 2 weeks and generally resolves at 3 to 4 weeks after the ischemic event. While important for clearing the tissue of compromised cells and debris and preparing it for transitioning into the proliferative phase of infarct healing, inflammation that becomes excessive or chronic results in adverse remodeling, infarct expansion, and poor patient outcomes. [1-4]

At the cellular level, resident cells including cardiomyocytes, endothelial cells, vascular smooth muscle cells, and fibroblasts that survive initial ischemia-induced necrosis are threatened by extracellular “danger” signals that may induce apoptosis. In response to the abundance of ROS and necrotic cell debris, they draw neutropils and later monocytes/macrophages to the injured area by releasing chemoattractants. Inflammation at the cellular level after MI is characterized by both harmful and helpful processes – continued cell death, release of inflammation-amplifying diffusible bioactive mediators and ROS, and clearing of dead cells, cellular debris, and disrupted ECM and finally release of pro-resolution “stop signals” and anti-inflammatory mediators. [2,4]

Since myocardium generally has only very limited regenerative capability, the space vacated by the death of cardiomyocytes is filled by a fibrotic scar. Therapeutic strategies targeting the cells affected by MI (i.e., resident cells) and the cells responding to MI (i.e., invading cells) offer unique opportunities to influence patient outcomes. New research into the promotion of myocardial regeneration using cell-based therapy strategies, such as stem cell transplantation or stem cell mobilization, appears promising. However, so far most of the positive effects of stem cells appear to result from paracrine actions of the cells on existing tissues rather than differentiation and incorporation of these stem cells at the lesion site. Paracrine signaling of engrafted cells may act by reducing apoptosis, inflammation, and fibrosis, and encouraging angiogenesis and other repair processes. [1,2] At the cellular level, several pharmacological agents are being evaluated for their therapeutic benefits after acute MI. These are generally acting either to directly protect resident cells or dampen the actions of infiltrating cells. Adrenomedullin has demonstrated cardoprotective effects in experimental animal models of I/R injury including suppression of cardiomyocyte apoptosis via interference with the PI3K-Akt cascade and inhibition of ROS production, resulting in reduced infarct size. In clinical studies, Adrenomedullin appears to reduce oxidative stress and reduce infarct size in patients undergoing reperfusion therapy. [5] Compounds that act on signaling molecules in the RISK (signaling pathway compromising the reperfusion-injury salvage kinase) pathway, such as Atrial Natriuretic Peptide (ANP), appear to have cardioprotective effects that include suppression of apoptosis and induction of pro-survival signaling pathways. In ex vivo animal hearts, ANP delivered during reperfusion decreases infarct size. In clinical studies, ANP administered to patients receiving reperfusion therapy appears to guard against ischemic reperfusion (I/R) injury and improve LV function. [5,6] Systemic immunosuppression achieved via drugs such as Methotrexate delivered in combination with reperfusion therapy have been shown to reduce infarct size in animal models. [1] Adenosine, when administered along with reperfusion therapy within 6 hours of MI, reduces infarct size and improves outcomes in experimental animal models and patients. Adenosine is known to inhibit neutrophils and platelets as well as dilate cardiac microvasculature and serve as a substrate for ATP replenishment. [5,6]

At the molecular level, an abundance of bioactive compounds act via autocrine, paracrine, and endocrine signaling to initiate signal transduction cascades that ultimately influence gene transcription. All of these interactions are regulated in space (i.e., different cell types) and time (i.e., after onset of infarct symptoms) to create an initially detrimental and eventually beneficial microenvironment after MI. Although these enormous complexities result in relatively slow progress toward effective therapies, there are innumerable opportunities for targeted therapeutic intervention.

MD Biosciences offers preclinical models for myocardial or I/R drug research. If you'd like to explore a study design, contact us and we'd be happy to discuss a protocol with you.

Steffens, S., Montecucco, F., and Mach, F. (2009). Thrombosis and Haemostasis, 102(2), 240-247.
Jiang, B. and Liao, R. (2010). Journal of Cardiovascular Translational Research, 3(4), 410-416.
Sun, Y. (2009). Cardiovascular Research, 81(3), 482-490.
Dobaczewski, M., Gonzalez-Quesada, C., and Frangogiannis, N.G. (2010). Journal of Molecular and Cellular Cardiology, 48(3), 504-511.
Yasuda, S. and Shimokawa, H. (2009). Circulation Journal, 73(11), 2000-2008.
Liem, D.A., Honda, H.M., Zhang, J., Woo, D., and Ping, P. (2007). Journal of Applied Physiology, 103(6), 2129-2136.

A preclinical model of post-operative pain that closely resembles human physiology, anatomy and metabolism

MD Biosciences — Mon, 23 Apr 2012 18:56:00 GMT

The management of post-operative pain is a challenge for both physicians and patients. In addition to a comfortable recovery, the prevention of chronic pain and improvement of conventional outcomes are important in post-operative pain management.

The rat plantar incisional model (brennan) has been a popular model for post-operative pain research. It offers researchers the ability to examine the wound healing process, local and systemic inflammatory responses, neurophysiological changes in primary afferents and dorsal horn neurons and te effects of potential new therapeutics. While the rat plantar incisional model continues to provide valuable data, the development and assessment of topical and local treatment methods in this model is limited.

Pigs are comparable to humans in a number of ways that make them a better choice than rodents when modeling particular aspects of post-operative pain. The skin of pigs is relatively hairless and is structurally similar to humans in terms of epidermal thickness, fixed nature of subcutaneous layer, patter of cutaneous blood flow and immune cells that localize to the skin. Pigs have become a standar model of the wound healing process because of similarities to the human wound healing including re-epithelialization rather than contraction. These qualities allow researchers to simultaneously examine wound healing and pain evaluation in order to gain a better understanding of interactions between the processes.

For this reason, we have developed a model of post-operative pain based on the benefits of the Brennan rat model but with a closer approximation to human physiology, anatomy and metabolism.

Benefits of the pig as a model of post-operational pain:

Duration and level of pain lasts at least 7 or more days post surgery, which enables possible assessment by repeated treatment for development of new analgesic agents
The location of the incision provides convenient application of patches, cream injections or other local treatment
Allows for follow-up monitoring of incision closure and healing
Responsive to local as well as systemic treatments
Similarities in pain tolerance between human and pigs
Analgesic responses to medications comparable between humans and pigs
Measurement of pain response via Von Frey, similar to that in humans

References

Sullivan, TO, Eaglestein WH, David SC, Mertz P: The pig as a model for human wound healing. Wound repair and regeneration 2001; 9:66-76
Marquest F., Bonneau M., Pascale F., Urien C., Kang C., Schwartz-Cornil I., Bertho N: Characterization of dendritic cells subpopulations in skin and afferent lymph in the swine model. PLoS One 2011; Jan 27:6(1):el6320
Swindle M., Makin A., Herron A., Clubb Jr F., and Frazier K: Swine as models in biomedical research and toxicology testing. Vet Pathol. 2012; Mar 49(2):344-56. Epub 2011 Mar 25.

Behavioral outcome measures in preclinical models of stroke

MD Biosciences — Thu, 19 Apr 2012 11:45:00 GMT

Preclinical stroke models are critical to our understanding of the mechanisms and neurological deficits following human stroke. While reducing infarct size is a focus of stroke therapies, much attention is also on neuroprotective properties. Adding behavioral and functional outcome measures to preclinical studies is important to evaluate the impact on impairments that occur following stroke: learning, memory, motor function and sensory. There are many behavior tests, each having different sensitivities to deficits associated with particular areas of brain damage.

The following chart below describes common preclinical models for global and focal cerebral ischemia, the expected area of damage in the brain, the relative cognitive and behavior tests and the histological assessment of the brain.

Table 1: Stroke Models
		MCAo (Focal Ischemia)		Multimodal models
	4VO (Global Ischemia)	Permanent	Transient	microsphere/ CLots
Expected area of damage	Mainly hippocampus	Cortex, stratum/Globus Plidus, thalamic nucleus, hippocampus	Mainly cortex	Cortex, multi-focal
Cognitive/ behavior tests	Spatial memory	Spatial memory, contralateral motor function, coordination	Contralateral motor function, coordination	Mainly motor function, spatial fuction
Histology Assessment of damage	H&E: cells at the level of hippocampus	Thionin/H&E: infarct size assessment	Thionin/H&E: infarct size assessment	Thionin/H&E: identification of infarct location & assessment of size

There are many behavior tests that can be added to preclinical models of stroke. Different tests are sensitive to deficits associated with particular areas of damage. For that reason, it is crucual to select models that provide damage in the relevant areas and pair them with functional tests that can complement histological data. In this way tests will be relevant to the location of damage and the extent of the damage. Contact a neurologist to discuss a preclinical study design.

We will continue to explore the various tests that can be added to assess cognitive, motor and sensory function in future posts. To read more about the neuroinflammation cascasde following acute ischemic stroke, download our eBook.

Modeling human pain in preclinical models - translating to clinic.

MD Biosciences — Fri, 30 Mar 2012 14:16:00 GMT

Management of acute pain related to surgical intervention, termed postoperative pain, continues to be a major problem facing physicians and patients today. The most common method for addressing post-operative pain is through pharmacotherapy. [1,2] Table 1 lists a selection of the most common analgesics used to treat acute surgical pain, their methods of delivery, and the mechanism by which they are thought to act. [1] Significant progress in the pain management field has been made in recent years mostly in the areas of new delivery methods and multimodal analgesia. Novel drug delivery systems for postoperative pain medications include, for example, patient-controlled analgesia, means of sustained or extended release, transdermal delivery using iontophoresis, and transmucosal and intranasal delivery systems. While a few of these methods may not yet be approved in all geographies, the majority now serve as new tools available to physicians to treat their surgical patients. [1,2] Multimodal analgesia is based on the idea that simultaneous administration of more than one pain therapy strategy offers opportunities for results that are either additive or synergistic. Although clinical data on these types of strategies are still somewhat inconsistent, some clinical trial data do demonstrate improved outcomes and reduced incidence of persistent post-operative pain. [1]

Commonly used pharmacotherapies for treating post-operative/surgical pain
Drug Type	Delivery Method	Probable Mechanism
Local anesthetics (bupivicaine, lidocaine)	EA/SA, PNB/C, SC, TR	Inhibition of sodium channel
Opioids (fentanyl, morphine)	EA/SA, IV, SC, TR	u-receptor agonist
Paracetamol	PO, IV	Uncertain
Non-steroidal anti-inflammatories (NSAIDs) (celecoxib, ibuprofen, keterolac)	PO, IV	Inhibition of cyclooxygenase
Gabapentinoids (gabapentin, pregabalin)	PO	Inhibition of voltage-gated sodium channels
alpha2-agonists (clonidine, dexmedetomidine)	PO, IV	alpha2-receptor agonist

Abbreviations: EA/SA = epideral/spinal, PNB/C = peripheral nerve block/catheter, SC = subcutaneous, TR = transdermal, IV = intravenous, PO = oral

Unfortunately, despite the critical nature of effective pain management after surgery and all of the tools available to physicians to address this issue, it is generally accepted that postoperative pain is undertreated. [1,2] Some estimates put adult patients receiving adequate postoperative pain relief at only 1 in 4 and children at roughly 1 in 5, numbers that clearly demonstrate the inadequacy of current strategies for postoperative pain management and the critical need for meaningful pain research that can be translated to the clinical setting. [1]

Relevant preclinical pain models for assessing post surgical pain are important. Modeling human pain in the animal setting and translating that to the clinic has certainly been a challenge. Recent innovative advances in preclinical models of post-operative pain are facilitating our understanding of its underlying mechanisms, furthering our efforts toward safe and effective therapeutic strategies of post-operative pain management.

MD Biosciences has developed a post-operative pain model that enables the evaluation of local treatments for post-operative or surgical pain. Pain lasts up to 14 days and evaluation of wound healing, pain assessments and inflammation can be assessed simultaneously. Ideal for analgesics that are injectables, implants, devices, patches and creams.

To learn more, download the whitepaper:

References

Wu, C.L. and Raja, S.N. (2011) Treatment of acute post-operative pain. The Lancet 377(9784):2215-2225.
Mao, J (2009) Translational pain research: achievements and challenges. Journal of Pain 10(10):1001-1011.

Toll-like receptor family member 4 (TLR4) in neuropathic pain

MD Biosciences — Mon, 19 Mar 2012 14:55:00 GMT

In the context of neuropathic pain (NP), toll-like receptor member 4 (TLR4) is known to be expressed exclusively on spinal microglia and significantly up-regulated upon peripheral nerve injury. TLR4-knockout mice display reduced effects of chronic chonstriction injury (CCI) induced nerve damage. Similary, TLR4 loss-of-function mutant mice as well as TLR4 antisense oligonucleotide-treated rats both display attenuated neuropathic pain symptoms after nerve damage. Further, intrathecal administration of a TLR4 antagonist after CCI treatment results in relief of neuropathic pain symptoms. Many exogenous and endogenous ligands are known to stimulate TLR4-mediated signaling. However, both in vitro and in vivo studies involving spinal nerve ligation (SNL) treated animals implicate Fibronectin in neuropathic pain-related TLR4 signaling. Fibronectin is an extracellular matrix protein that is commonly produced in response to tissue injury. When administered intrathecally to intact rats, Fibronectin induces microglial up-regulation of the purigenic receptor, P2X4, and symptoms of neuropathic pain. This stimulation of P2X4 expression can be suppressed by interuption of Fibronectin binding the TLR4 receptor after SNL injury in rats.

Download the rest of the eBook on 5 key pathways to microglial activation.

References

Milligan, E.D. and Watkins, L.R. (2009) Pathological and protective roles of glia in chronic pain. Nature Reviews Neuroscience 10(1):23-36.
Inoue, K and Tsuda, M (2009) Microglia and neuropathic pain. Glia 57(14):1469-1479.
Graeber, M.B. (2010) Changing face of microglia. Science 330(6005):783-788.
Smith, H.S. (2010) Activated microglia in nociception. Pain Physician 13(3):295-304.

Pain-related characteristics in MIA-induced preclinical model of OA

MD Biosciences — Fri, 09 Mar 2012 16:06:00 GMT

Osteoarthritis (OA) is a widespread condition that affects greater than 70% of the elderly population and poses a heavy cost burden on healthcare. It is a chronic degenerative disease characterized byt the loss of articular cartilage components, which affects the entire joint structure. One of the major complaints by OA patients is the loss of joint function as well as chronic pain. Current therapies are focused on alleviating joint pain, however full pain relief is rarely experienced and significant side affects are commonly present. Research is not only focused disease pathology but also on understanding the mechanisms responsible for induction and maintenance of pain states.

The monosodium iodoacetate (MIA) model has been well characterized for the evaluation of OA pain. The iodacetate creates local inflammation in the knee from fluid expansion of the synovial mebrane, which is then followed by the production of inflammatory mediators (TNF-a, IL-1b, IL-6 and NGF). These mediators further contribute to OA pathogenesis by increasing cartilage degradation. The cartilage damage and degradation leads to chronic neuronal damage with neuropathic characteristics. The MIA-OA model then is thought to contain both inflammatory and neuropathic pain-related states by using local tissues and sensory innervation of the peripheral nervous system.

Inflammatory-related Pain State

CGRP (calcitonin gene-related peptide) is elevated in the sensory neurons innervating the knee and DRG (dorsal root ganglia). CGRP is an inflammatory driven neuronal marker whose expression is dependant on NGF (nerve growth factor) expression. The elevation of CGRP produces hyperalgesia and a pain sensation derived from inflammation.

Neuropathic-related Pain State

It is also suggested that proliferation of microglia in the dorsal horn of the spinal cord along with elevated levels of pro-inflammatory cytokines contributes to neuronal damage. The neuronal damage gradually rises along with the progress cartilage damage and this may lead to the chronic neuropathic pain state.

MIA-induced OA model

The MIA-induced OA model can allow the evaluation of anti-inflammatories (to treat early symptoms) as well as analgesics (to treat chronic pain). The study can be run anywhere from 14 days out to 56 days or longer depending on the proposed therapy.

Readouts:

Weight bearing measurements: weight bearing measures the spontaneous pain as a result of nerve injury or inflammation.
Open field: the open field test would measure the animals desire to walk, the distance and speed if they did walk.
Rotorod: the rotorod test would measure the ability to withstand the pain in a situation where walking was required.
Joint compression: Randall Selitto can be used to measure the pressure required to withdraw (compression threshold)
Histology and xray analysis of the knee
Biomarkers (mRNA and protein level)

Weight bearing measurements (% of weight carried on ipsilateral leg) for gabapentin and vehicle groups in the MIA-induced OA model.

Weight bearing (% difference between left and right leg) of gapabentin and vehicle control in the MIA-induced model of OA.

Discuss a study outline with a scientist by contacting us.

References

Orita, et al. (2011) Pain-related sensory inervation in monoiodoacetate-induced osteoarthritis in rat knees that gradually develops neuronal injury in addition to inflammatory pain. BMC Musculoskeletal Disorders 12:134
Harvey, V and A. Dickenson (2009) Behavioral and electrophysiological characterisation of experiementally induced osteoarthritis and neuropathy in C57BL/6 mice. Molecular Pain 5:18

Preclinical models for ischemic stroke

MD Biosciences — Tue, 28 Feb 2012 17:15:00 GMT

There are currently a large number of well-characterized, ischemic stroke animal models available for pre-clinical research. These models can be categorized into those two groups – those for the study of stroke-associated risk factors and those for the study of stroke pathophysiology. The latter can be further separated into models of focal verses global ischemia and are listed:[1]

Focal Ischemia Models

Middle Cerebral Artery occlusion (MCAo)
Thromboembolic MCA occlusion
Endothelin model
Photothrombosis model

Global Ischemia Models

Four vessel occlusion (4VO)
Two vessel occlusion (2VO)
Cardiac arrest and resuscitation

The most commonly employed model is that of middle cerebral artery occlusion (MCAO) in rats. MCAO is generally achieved mechanically and may be either transient with variable duration or permanent. It is highly reproducible, requires minimal surgical intervention, and produces predictable infarcts similar to clinical observations. Rats offer the advantages of being small enough to allow sufficient numbers to be included in studies without increased costs and yet large enough to allow monitoring of physiological variables during experiments. [1] However, the main disadvantage is that the brains of rats are quite dissimilar to humans in many aspects including of size, gross anatomy, neuroanatomical connectivity, cognitive ability, and cerebral vasculature. Further, young and healthy rats are typically used, which do not adequately represent the demographic of humans who tend to experience acute ischemic stroke – those with risk factors that include advanced age, cardiovascular disease, diabetes, and others. This is perhaps one reason why promising, new neuroprotective strategies developed in the pre-clinical setting often fail in clinical trials, emphasizing the fact that care must be taken when interpreting data and translating findings to the clinical setting. [1, 2]

Despite these obstacles, our understanding of post-ischemic stroke neuroinflammation has advanced significantly with the help of in vitro systems and the MCAo, 4VO and other in vivo models and lessons taken from failed pre-clinical and clinical trials and autopsy results.

MD Biosciences offers MCAo and 4VO models for ischemic stroke. If you'd like to speak with a scientist regarding these models, please contact us.

Ceulemans, A.-G., Zgavc, T., Kooijman, R., Hachimi-Idrissi, S., Sarre, S., and Michotte, Y. (2010). The dual role of the neuroinflammatory response after ischemic stroke: modulatory effects of hypothermia. Journal of Neuroinflammation 7:74.
Bacigaluppi, M., Comi, G. and Hermann, D.M. (2010). Animal models of ischemic stroke. Part two: Modeling cerebral ischemia. The Open Neurology Journal 4:34-38.

Inflammation after acute ischemic stroke- preclinical models

MD Biosciences — Tue, 14 Feb 2012 21:00:00 GMT

The most common form of stroke is acute ischemic stroke (approximately 85% of cases), which is caused by either an atherothrombosis in a major cervical or intracranial artery or an embolism that travels from the heart. The resulting occlusion causes a sudden deficiency of oxygen and glucose in the brain region normally serviced by the blocked artery. Victims of large-vessel ischemic strokes lose on the order of 100 million neurons per hour prior to treatment, causing immediate, permanent neural damage in the infarct area, termed the ischemic core. Further neural damage occurs in the areas surrounding the core, called the penumbra, where the tissue becomes highly inflamed and slowly dies. Stroke sufferers experience a range of neurological deficits including partial paralysis, impaired memory, loss of speech, and/or decreased cognition and many become permanently disabled, requiring institutional care. [1-4]

Unfortunately, current stroke therapies approved for human use are very limited. The only drug clinically available in the US is intravenous recombinant tissue plasminogen activator (rt-PA), a thrombolytic agent that has been shown to improve stroke patient functional outcomes. However, rt-PA is only effective if administered within the first 3 hours after symptom onset and carries with it a significant risk of intracranial hemorrhage. Consequently, only about 5 to 10% of patients can receive this therapy. Over 1,000 other potential stroke therapeutics have been tested in preclinical cerebral stroke models and approximately one tenth of these have made it to clinical trials. However, the majority of these efforts have already failed. It is likely that the most effective way to improve outcomes is rapid reperfusion of the ischemic area using thrombolytic means in combination with neuroprotective strategies to salvage cells within the penumbra and prevent them from becoming part of the ischemic core. To this end, researchers have been increasingly focused on post-stroke neuroinflammation and the role it plays in neurotoxicity and neuroprotection. [1-4]

References

Candelario-Jalil, E. (2009). Injury and repair mechanisms in ischemic stroke: considerations for the development of novel neurotherapeutics. Current Opinion in Investigational Drugs 10(7):644-654.
Ceulemans, A.-G., Zgavc, T., Kooijman, R., Hachimi-Idrissi, S., Sarre, S., and Michotte, Y. (2010). The dual role of the neuroinflammatory response after ischemic stroke: modulatory effects of hypothermia. Journal of Neuroinflammation 7:74.
Downes, C.E. and Crank, P.J. (2010). Neural injury following stroke: are Toll-like receptors the link between the immune system and the CNS? British Journal of Pharmacology 160:1872-1888.
Lakhan, S.E., Kirchgessner, A., and Hofer, M. (2009). Inflammatory mechanisms in ischemic stroke: therapeutic approaches. Journal of Translational Medicine 7:97.
Bacigaluppi, M., Comi, G. and Hermann, D.M. (2010). Animal models of ischemic stroke. Part two: Modeling cerebral ischemia. The Open Neurology Journal 4:34-38.

Microglial involvement in Neuropathic Pain: 5 activation pathways

MD Biosciences — Wed, 26 Oct 2011 14:25:00 GMT

Neuroinflammation is a common thread in neuropathic pain (NP), regardless of the conditions under which neuropathic pain develops. This opens up a whole new avenue for investigations into neuropathic pain pathology. Since the primary cell type responsible for immune-like functions in the CNS is microglia, many researchers have turned their attention toward working to better understand microglial physiology and its potential involvement in neuropathic pain.

[short overview of microglial cells can be found here]

Microglial participation in NP pathophysiology has been investigated using a wide variety of experimental preclinical models. Some of the most common models used are the CCI, SNL and STZ-induced Diabetic neuropathy model. Several lines of evidence compiled using these models have demonstrated the intimate involvement of microglial cells in the establishment of neuropathic pain. More specifically, the process of microglial activation is now thought to be both necessary and sufficient for neuropathic pain initiation. Although there is some variability between results obatined using the different neuropathic pain models, generally microglial cells in the ipsilateral dorsal horn of the spinal cord become activated within approximately 4 hours, increase 2- to 4-fold in number by day 2 and remain active for several months after peripheral nerve injury. These effects can be suppressed by non-specific microglial inhibitors in these preclinical models. In the context of neuropathic pain, local, responding microglial cells are known to be activated by a broad range of stimuli, five predominant activation pathways appear to be most critical and are identified by their major ligand receptor.

TLR4 (toll-like receptor family member 4)
P2X4 (purinociceptor 4)
INF-g and CB2
MCP-1
Fractalkine

These mechanisms have emerged as exciting new focal points for assessing opportunities for the future development of pharmacotherapies, gene therapies or cell-based therapies for neuropathic patients. You can read more about the activation pathways in our new eBook.

Overview of microglial cells in the CNS

MD Biosciences — Mon, 24 Oct 2011 20:56:00 GMT

Of the roughly 70% of cells in the central nervous system (CNS) that are glia, appromixately 5-10% are microglial cells. Microglial cells are derived from peripheral myeloid progenitor cells that enter the CNS during embryonic development. Though ubiquitous in the CNS, microglial cell densities vary by region. They function to provide structural and trophic support to neurons and serve as the resident immune-competent cells of the CNS, tasked with:

detection of infections and injuries
protection of healthy tissues
elimination of disturbances
restoration of homeostatic conditions

Normally, microglial morphology is characterized by small soma with many thin, branded processes. Microglial processes come in contact with neurons, endothelial cells and astrocytes but not other microglial cells. In fact, each cell appears to be responsible for a distinct territory, within which it contantly samples the extracellular microenvironment by sweeping its processes through the tissue without disrupting neuronal connectivity.

Microglial cells have a very low threshold for activation and can be activated by a wide vavriety of stimuli. Once activated, they undergo morphological and phyiological changes and they mobilize and proliferate. Activated cells display enlarged soma with shorter processes or even amoeba-like shapes and drmatically altered gene expression profiles. They home to injured areas, perform phagocytic and antigen presentation functions, and re-enter the cell cycle to increase their number. As microglial cells are not electrically coupled with other cells, they act solely via the release of diffusible mediators to communicate with neighboring cells in a paracrine fashion. Microglial phenotypes are extremely plastic. The process of microglial activation is neither an "all-or-none" committment, nor a linear path, which allows for creation of a wide range of activated phenotypes to achieve very graded responses to real or perceived threats to the CNS. Taken together with evidence of microglial populations haven already "built-in" heterogeneity and the possibilitiy that when individual cells are activated once, they may respond differently when activated again through potentially long-lasting epigenetic mechanismsm, the picture of microglial activities in the CNS becomes extremely complex.

Behavior based findings in preclinical nerve injury models

MD Biosciences — Wed, 24 Aug 2011 20:00:00 GMT

We get a lot of questions on the various neuropathic pain models and how to choose the one that's most appropriate or a comparison of what's involved with each model (e.g. surgery, behaviors, centralization, peripheral vs central involvement etc). We thought it may be helpful to discuss the various aspects of these models to assist with the selection and understanding of the mechanisms and behaviors. Of course, it ultimately depends on the drug target and the pathway involved and we can certainly discuss individual specifics with you.

Neuropathic Pain Models

Preclinical models in pain research offer great promise for both the identification of pain mechanism and the investigation of possible therapeutic applications. Since there is not one mechanism that is responsible for generation and maintenance on neuropathic pain (NPP), it is essential to select the best model for each specific research interest or a combination of appropriate but distinct models.

At present, preclinical models for NPP cover various etiologies and are related to symptoms leading to an extensive picture of clinical NPP manifestations. The majority of these preclinical models of NPP involve traumatic injuries to peripheral nerves, nerve roots or spinal cord by transaction, ligation or compression (Fig 1). Other models are related to direct or indirect nerve inflammation, ischemia, drug toxicity or systemic metabolic disorder leading to nerve ending damage.

All of these models have been characterized by precise behavior-based evaluation using different methods of sensory stimulation. Many molecular, physiological and structural modifications have been described in these models. Most of the rodent models are described also with actual electrophysiology measurement and imaging techniques as well as genomic and proteomic screening. Over the coming weeks, we will discuss these various findings in nerve injury models.

Behavior based findings in two common preclinical Nerve Injury Models

Spinal Nerve Ligation (SNL)

Surgical procedures on selective spinal nerves such as the SNL model allows the direct access to sensory and motor fibers and clear segment location of injured (L5/L6) and non injured DRG (L4) and afferents. In this model the tight ligation of the L5/L6 spinal nerves results in robust and consistent sympathetic related neuropathic pain behavior including indirect signs of spontaneous pain, heat hyperalgesia, mechnical allodynia and cold allodynia.

Chronic constriction injury (CCI, also Bennet and Xie)

The CCI model is induced by applying 4 catgut loosely around the sciatic nerve. This model allows sensory testing in the hind paw as not all the sensory nerved are damages. Moderate autotomy, guarding and excessive grooming of the injured limb are reported as well as thermal hyperalgesia and mechanical allodynia were recorded. The main challenges in this model is the standardization of the loose but constrictive ligature.

The following table summarizes the behavior based response following each of the surgical-induced preclinical neuropathic pain models.

Parameter	CST	TST	CCI	SNL	PSL
Autotomy	65%	-	~10%	~10%	-
Natural Pain Behavior	Low	0	Significant peak at day 9	Moderate Peak at day 16	Low
Response to hot plate (duration of lifting time)	Low	Low	Significant, yet reversable with a peak at days 3-9 post surgery	Moderate persistance until day 28	Minimal only at day 3
Response to VF	Significant	Significant	Moderate	Significantly higher than other models	Significant
Pin Prick	Significant	Significant	Significant	Significantly higher than other models	Significant
Acetone Test	Significant starting from day 1	Significant starting from day 1	Significant starting from day 1	Significant starting from day 1	Significant starting from day 1
Cold Plate	Moderate from day 21	Minor from day 21	Significant from day 1	Moderate from day 1	Significant peak at day 14

Abbreviations: CST - complete sciatic transection; TST - tibial and sural transection; CCI - chronic constriction injury; SNL - spinal nerve ligation; PSL - partial nerve ligation; VF - von frey

Hopefully this comparison of the behavioral findings for each spinal nerve injury model helps sort out the differences and similarities between models. If you would like to speak more about the models as it relates specifically to your compound, please contact us. Our scientists love talking about this stuff.

New eBook: The link b/t pain & inflammation, targets in the overlap?

MD Biosciences — Tue, 19 Jul 2011 17:34:00 GMT

Neuropathic pain presents a wide variety of challenges to researchers, not the least of which is the simple fact that neuropathic pain, by definition, requires neuronal damage, which in turn automatically initiates immune response that often inflicts further neuronal damage. The interactions between the nervous system and immune system in the case of neuropathic pain make for a very complex story that is only beginning to unfold:

At the anatomical-level, neuro-immune interactions have been shown to take place all along the pain processing pathway. This is partially facilitated by increased permeability of the blood-brain barrier following SCI or peripheral nerve injury.

At the cellular level, neuro-immune interactions involve a variety of cells including mast cells, neutrophils, macrophages and T cells as well as glial cells with immune-like functions.

At the biochemical level, factors either directly produced by involved leukocytes and immune system factors released by glial cells expose prominent potenital therapeutic targets.

As neuroimmunologists find interactions between the nervous and immune systems, well-kown disorders may be found in the overlap. This eBook explores the immune system, inflammation, pain processing as well the various cells involved in the neuro-inflammation aspect of neuropathic pain and the potential inflammation-related drug targets.

Download the complimentary eBook: The Link between Pain and Inflammation

The ideal preclinical model system: large vs small species.

MD Biosciences — Wed, 08 Jun 2011 14:46:00 GMT

Common models for preclinical efficacy often use rodents as they are readily available, cost effective, easy to handle and most familiar to investigators. In choosing a preclinical model, one also needs to consider the anatomical/functional similarity to humans and there are cases where moving onto a larger species is more relevant to the clinic and human condition. Two of those cases are described below:

Post-operative pain and wound healing:

Small animals such as the rat and mouse different from humans in that they have a dense layer of hair on the body, a thin epidermis and they heal primarily through wound contraction as opposed to re-epithelization as in the swine and human. Anatomically and physiologically, swine skin is more similar to human skin:

Skin has thick epidermis
Well developed rete-ridges, dermal papillary godies and adundant subdermal adipose tissue
Swine dermal collagen is biochemically similar to human dermal collagen
Size, orientation and distribution of blood vessels in the dermis or porcine skin is similar to human skin
Sparse body hair which progresses through the hair cycle independently of neighboring follicles, which is importnat since they play a role in re-epithelialization
Overall physiology of porcine is sinmilar to human physiology with most organ systems being similar in anatomy and funtion

Acute myocardial infarct (AMI) and ischemic reperfusion (IR) injury

Anatomically, swine hearts are very similar in size and gross structure to human hearts and at the level of coronary vasculature are nearly identical - blood supply is right side dominat and pre-formed collaterals are absent. Physiologically, the baseline heart rate and blood pressure of swine are similar to humans.

AMI induction in swine is relatively easy by a variety of means and produces infarcts similar to those observed in humans with predictable sizes, locations and time courses. All cardioprotective schemes so far identified for humans have been described in swine after ischemia and reperfusion, namely hibernation ad ischemic pre- and post-conditioning.

Overall advantages

Experimentally, porcine are capable of tolerating long and complex protocols, medical device implantation and repeated surgeries. In the post-operative pain model, pain can be evaluated up to 12 days and wound healing/inflammation can be observed simultaneously with pain. In the cardiac models, hearts are large enough to allow myocardial biopsies to be taken both from infarct area and an unaffected area, providing a conventional internal control. Additionally, intracoronary drug delivery and implantation of devices or microdialysis probes enables measurement of small, diffusible bioactive molecules.

For information on either the post-operative pain model or acute myocardial infarct in swine, download the following whitepapers:

		Myocardial Infarct Models: Evaluating the myocardial protection of potential drug therapies or devices.
		A model of post-operative pain: assessment of analgesic affects of local treatment strategies.

References

Sullivan, T.P. et al., (2001) Wound Rep Reg. 9:66
Klocke, R., Tian, W. Kuhlmann, M.T., and Nikol, S. (2007). Cardiovascular Research, 74, 29‐38.
Dixon, J.A. and Spinale, F.G. (2009). Circulation: Heart Failure, 2(3), 262‐271.
Swindle, M.M., Makin, A., Herron, A.J., Clubb, F.J., and Frazier, K.S. (2011). Veterinary Patholology, Mar 25 [Epub ahead of print].
Heusch, G., Skyschally, A., and Schulz, R. (2011). Journal of Molecular and Cellular Cardiology, Mar 5 [Epub ahead of print].

Receptor & Receptor Ion Channels involved in neuropathic pain.

MD Biosciences — Tue, 07 Jun 2011 14:58:00 GMT

This post continues on our discussion of potential inflammation-related drug targets for the treatment of neuropathic pain. See also Pro-inflammatory cytokines and anti-inflammatory cytokines as targets in neuropathic pain.

TLR4 and its relevance to neuropathic pain

Toll-like receptors (TLRs) are a family of 13 pattern recognition receptors expressed by leukocytes that are responsible for identifying foreign toxins and microbes and initiating inflammation as a part of the innate immune response. TLR4 is expressed on macrophages, microglia and Schwann cells.

TLR4 is thought to be activated by necrotic cells, injured axons, and extracellular matrix components. Elimination or modification of TLR4 function at either the receptor itself (via knockout, point mutation, antisense oligoneucleotide or antagonist treatment) or its associated signal transduction cascade reduces or completely prevents microglial activation and associated cytokine release. This leads to further macrophage recruitment, microglial activation, pain hypersensitivity, hyperalgesia and allodynia.

P2X4R and its relevance to neuropathic pain

Purigenic receptors are a large family of receptors that bind various forms of adenosine nucleotides. The P2X subtype receptors (of which there are 7 currently known) are extracellular ARP- or ADP-sensitive ligand-gated ion channels found on a variety of neuronal and glial cell types.

P2X4R expression is up-regulated in spinal cord microglia upon nerve damage, the inhibition of which prevents allodynia. Further, intrathecal application of ATP activates microglia and intiates allodynia in rats. Pharmacological inhibition of P2X4R reduces allodynia and lack of P2X4R prevents allodynia development after nerve damage. Presumably, the mechanism by which P2X4 is functioning in the development of hyperalgesia and allodynia in animals models of neuropathic pain involves ATP released from activated astrocytes.

TRPV1 (vanilloid receptor) and its relevance to neuropathic pain

TRPV1 is a ligand-gated ion channel expressed in nociceptors and is a member of the transient receptor potential (TRP) family of ion channels. TRPV1 can be activated by capsaicin, low pH, nixious heat, spider toxins, and the endocannabinoid, AEA.

The expression and function of TRPV1 is altered under inflammatory conditions by a variety of mechanisms. Inflammatory mediators including TNF, PGE2, and Bradykinin alter expression of TRPV1 in nociceptor cell bodies in the DRG, trafficking of TRPV1 to peripheral terminals, and activity of TRPV1 once inserted in the membrane. In models of neuropathic pain, TRPV1 antagonists reduce pain hypersensitivity.

Choosing the appropriate neuropathic pain model is dependent upon the target and mechanism of the compound. For further information on neuropathic pain models, download the whitepaper: Periphery Nerve Injury Models: Understanding underlying mechanisms of neuropathic pain.

References

Austin, P.J. and Moalem-Taylor, G (2010) Nociceptors: the sensors of the pain pathway. Journal of Clinical Investigation. 120(11):823
Smith, H.S. (2010) Activated microglia in nociception. Pain Physician. 13:295
Leung, L and Cahill, C.M. (2010) TNF-alpha and neuropathic pain - a review. Journal of Neuroinflammation. 7(1):27
Stein, C. et al. (2009) Peripheral mechanisms of pain and analgesia. Brain Research Reviews. 60(1):90
Schlosburg, J.E. et al., (2009) Targetting fatty acide amid hydrolase (FAAH) to treat pain and inflammation. The American Association of Pharmaceutical Scientists Journal 11(1):39
Patapoutian, A. et al., (2009) Transient receptor potential channels: targeting pain at the source. Nature Reviews Drug Discovery. 8(1):55

Preclinical contact hypersensitivity models - DNCB or FITC?

MD Biosciences — Wed, 01 Jun 2011 14:37:00 GMT

Contact hypersensitivity dermatitis occurs when the immune system mounts a response to chemicals the body comes into contact with via the skin. Alone these chemicals would be too small for the immune system to respond to, but they are all capable of binding to proteins within the body, a process termed haptenation. In most individuals this is harmless however in some individuals an immune response against chemicals bound to self proteins is mounted leading to inflammation of the skin at the contact site. Many commonly encountered chemicals are capable of acting as contact sensitizers, these include petrochemicals, heavy metal ions (i.e Nickel) and some plant extracts (i.e urushiol from poison ivy).

DNCB-induced Contact Dermatitis

The 2,4-Dinitrochlorobenzene (DNCB) induced contact dermatitis model is Th1 mediated with IFN-g production by both CD4+ and CD8+ cells observed and increased IL-12p40 mRNA observed in the draining lymph node. Cell mediated responses are thought to be of particular importance in the pathology associated with challenge of sensitised individuals.

FITC-induced Contact Dermatitis

The Fluorescein isothyocyanate (FITC) induced contact dermatitis model is mediated by the Th2 pathway. Unlike many contact hypersensitivity reactions which induce strongly cytotoxic T cell mediated responses; the response to FITC challenge exhibits many of the hallmarks of atopic dermatitis; Local eosinophilia, mast cells infiltration, Anti-FITC IgE and IL-4 and IL-10 production by CD4+ cells are observed following sensitisation. In addition immediate and late phase responses are observed. Work to dissect the pathological mechanism has taken place, yielding the following results; The passive transfer of immune sera results in a rapid transient response to FITC challenge (peaking 15-30 minutes, returning to normal by 24hrs), The adoptive transfer of LN cells from sensitised mice results in a more delayed and sustained ear swelling, The depletion of CD4+ cells prior to adoptive transfer prevents ear swelling following application of FITC. These results suggest that IgE is responsible for the immediate phase of the response to FITC application while CD4+ cells sustain the response.

It is becoming apparent that a role for the Th17 pathway may also be important in the development of contact hypersensitivity and allergic responses, and has been implicated in the FITC induced contact dermatitis model. The Aryl hydrocarbon receptor (AhR) activation is a cofactor in the development of Th17 responses and AhR null mice have been shown have impaired Langerhans cell maturation and as a result impaired responses to FITC sensitisation.

Learn more about the DNCB and FITC-induced Contact Dermatitis preclinical efficacy models.

References:

Dearman RJ, and Kimber I. Role of CD4(+) T helper 2-type cells in cutaneous inflammatory responses induced by fluorescein isothiocyanate. Immunology. 2000;101(4):442-51.
Dearman RJ, Humphreys N, Skinner RA, and Kimber I. Allergen-induced cytokine phenotypes in mice: role of CD4 and CD8 T cell populations. Clin Exp Allergy. 2005;35(4):498-505.
Takeshita K, Yamasaki T, Akira S, Gantner F, and Bacon KB. Essential role of MHC II-independent CD4+ T cells, IL-4 and STAT6 in contact hypersensitivity induced by fluorescein isothiocyanate in the mouse. Int Immunol. 2004:16(5):685-95.
Hayashi M, Higashi K, Kato H, Kaneko H. Assessment of preferential Th1 or Th2 induction by low-molecular-weight compounds using a reverse transcription-polymerase chain reaction method: comparison of two mouse strains, C57BL/6 and BALB/c. Toxicol Appl Pharmacol. 2001:177(1):38-45.
Cowden JM, Zhang M, Dunford PJ, and Thurmond RL. The Histamine H4 Receptor Mediates Inflammation and Pruritus in Th2-Dependent Dermal Inflammation. J. Inv. Dermatology 2010: 130, 1023–1033.
Jux B, Kadow S, and Esser C. Langerhans Cell Maturation and Contact Hypersensitivity Are Impaired in Aryl Hydrocarbon Receptor-Null Mice. J Immunol. 2009;182;6709-6717.

Inflammatory events underlying cardiovascular disease.

MD Biosciences — Tue, 10 May 2011 19:51:00 GMT

Cardiovascular disease (CVD) including heart disease, vascular disease and atherosclerosis are the most critical global health threats.

An estimated 26 million people are living with the effects of heart disease and is a major cause of death in western society. Until recently the widely held belief was that the CVD is simply the process as a build up of fat on the surface of artery walls. Eventually, this build up of fat blocks the artery and a heart attack or stroke occurs. However, the process has now been identified as a disease of the inner artery wall (intima) and inflammation is a key factor in its progression.

The source of inflammation in CVD is not completely understood. However, numerous factors are thought to initiate the complex inflammatory process such infectious agents for example herpes viruses and Chlamydia pneumoniae. Other promoters and stimulators of inflammation leading to endothelial injury include smoking, hyperglycaemia, oxidised low-density lipoprotein (LDL) or sheer stress on the vessel wall by hypertension. Genetic factors may also play a role in the degree and duration of the inflammatory response, although this still needs to be fully explored.

Once stimulated by a promoter or stimulator (including those mentioned above), endothelial cells of the intima interpret their presence as unwanted and activate the immune system to deal with the problem. The gene transcription factor NF-kB is released, serving as a promoter of early cytokines such as TNF-α and IL-6, chemokines such as MCP-1 and adhesion molecules. The chemokines attract monocytes and T lymphocytes (T cells) from the blood stream allowing monocytes to travel across the endothelial barrier and become macrophages. Entry of monocytes into the vessel wall is a key factor in the development of atherosclerosis, as blocking monocyte migration has ameliorated atherosclerosis in in vivo models (1). Once inside the intima, these mononuclear cells produce pro-inflammatory cytokines such as IL-1, IL-6 and TNF-α to stimulate the inflammatory cascade. Metalloproteinases are also released, promoting smooth muscle cell proliferation and uptake of LDL by these macrophages to form foam cells.

Through uptake of LDLs, a fatty streak can develop into a necrotic plaque that is sealed off from the blood flow by the fibrous cap and is held in balance by collagen deposition and degredation. Fissuring or rupturing of this cap can occur when the balance is disrupted by increased inflammation leading to thinning of the collagen cap. The plaque rupture exposes thrombotic substances to the blood, leading to local thrombus formation and downstream microemobolization (2). Furthermore, inflammatory cytokines activate platelets expressing P-selectin and CD40, thus increasing platelet-platelet adhesiveness (3). Cytokines also signal the production of acute phase proteins such as fibrinogen serum amyloid A and C-reactive protein. These are systemic downstream markers which can be useful in assessing cardiovascular risk in patients.

The role of inflammation in cardiovascular disease is not strictly limited to the innate inflammatory response. The adaptive immune response particularly lymphocytes are also involved in CVD. Flow cytometry based methods have quantitatively investigated the cell composition of a normal aortas (4, 5). These have demonstrated that both T and B lymphocytes, macrophages and dendritic cells reside within a major site of the arterial wall (lamina adventitia) of non inflamed aortas. To further visualise the induction of the immune response and investigate the relationship between the immune and cardiovascular systems, multiphoton laser-scanning microscopy (MPLSM) could be used, however this is still at a method development stage (6).

Prevention of the initial development of CVD and progression over time is the goal of any prevention program. With increasing knowledge, the approach to identifying the underlying causes of heart disease is changing rapidly. Much research has identified inflammation as an underlying or active factor in the development of the disease. For the past two decades, clinical trials of antiatherosclerotic drug therapies have sought to reduce CVD morbidity and mortality. This includes the use of a group of drugs called statins (atorvastatin and rosuvastatin) (7) to treat high cholesterol levels which have been shown in large randomised trials, to reduce cardiovascular events in risk patients (8). Research has demonstrated that at higher doses, statins slow or even reverse plaque progression as demonstrated during intravascular ultrasound (9). Recently however, clinical findings have indicated that statins may slow progression of disease at a rate and to an extent that cannot be attributed to lower LDL alone. The proposed mechanisms for such pleiotropic actions include endothelial-dependent nitric oxide bioavailability, inhibition of oxidative stress and anti-inflammatory activity. In particular a number of clinical trials have shown that statins reproducibly lower circulating levels of C reactive protein (CRP) an inflammatory biomarker associated with acute coronary syndromes (10). Reducing inflammation may therefore be a key mechanism by which statins alter the biology of the plaque and slow down disease progression.

Although statins are currently the most popular and widely prescribed drugs to help treat CVD, evidence indicates side effects such as a higher risk of drug interactions in elderly, muscle pain or memory related problems are linked to their use. It is therefore necessary to continue the investigation into inflammation and in inflammatory cell-cell interactions to help develop more effective therapies.

References

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Inflammation & Pain processing: Relevant preclinical efficacy models

MD Biosciences — Wed, 04 May 2011 18:46:00 GMT

Chronic, destructive inflammation is at the core of a wide variety of diseases and conditions.

Inflammation, whether acute or chronic, is very often associated with pain. Similar to inflammation, pain can be physiological (an adaptive means of protecting tissues from real or perceived danger) or pathological (chronic, and often debilitating despite resolution of the original stimulus). Chronic pain can be caused by a variety of situations including inflammatory diseases such as osteo‐ and rheumatoid arthritis (inflammatory pain), tumor formation (cancer pain), and nerve injury (neuropathic pain).

Pain Processing

While the process of physiological nociception and pain perception is very complex, depending on the quality, intensity, and locality of the stimulus and the species, developmental age, and psychological state of the subjects (i.e., stress level, anticipation, emotional state, etc.), the general pathway for transmitting pain information to the brain is well documented. Nociceptors are pseudounipolar neurons with unencapsulated peripheral terminals the skin, muscles, joints, or viscera; cell bodies residing in the dorsal root ganglion (DRG); and central terminals in the dorsal horn of the spinal cord. There are generally two types of nociceptors – A‐fibers are fast‐conducting with myelinated axons and have small receptive fields for stimulus localization while C‐fibers are slower with unmyelinated axons that are bundled into fascicles wrapped by Schwann cells and have broad receptive fields. Nociceptors normally are electrically silent and have a high threshold compared to somatosensory neurons involved in, for example, vision or hearing. Once stimulated, nociceptors produce all or nothing action potentials releasing glutamate as their primary neurotransmitter and having excitatory effects on postsynaptic cells in the dorsal horn. In the dorsal horn, primary afferent neurons either synapse directly with projection neurons or, more commonly, first with a variety of excitatory and inhibitory interneurons for signal modification. Ascending projection neurons extend, mostly contralaterally, to supraspinal targets including the caudal ventrolateral medulla, the nucleus of the solitary tract, the lateral parabrachial area, the periaqueductal grey matter, and the thalamus. Descending pathways projecting from the nucleus raphe magnus and the locus coeruleus release serotonin and norepenephrin, respectively, via volume transmission in the DRG to further modify pain processing. All along the pain processing pathway, from the primary afferent nociceptors, to the dorsal horn of the spinal cord, to the supraspinal processing centers and including descending projections that further modify processing, there is a delicate balance of excitation and inhibition that is important for properly representing the pain stimulus. Miss‐communication at any of these locations can result in chronic pain.

Selecting Relevant Preclinical Models

Pain therapies can provide relief either through targeting sensitizing agents or by inhibiting the activity of neurons involved in the pain processing directly. Choosing the appropriate pain model should be based off the primary mechanism, site of action, drug class, and required behavioral readouts. Additionally, pain models themselves can be highly customized once the appropriate model has been selected based on the mode of delivery and target. MD Biosciences has extensive experience working with a wide range of drug classes as well as customized applications for route of delivery. We can help choose the appropriate model and approach for your pain therapeutics program. Read a case study covering customized approaches in pain therapies and contact us if you would like to discuss your program.

The link between TH17 & osteoclast function in RA

MD Biosciences — Tue, 19 Apr 2011 16:33:00 GMT

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease that affects approximately 1% of the population, and in 2010 cost the US alone $39.2 billion (1,2). The disease is characterized by bone erosion, cartilage damage, synovial hyperplasia and cellular infiltration, all of which result in debilitating joint pain and stiffness (1,3,4). Studying preclinical models such as the collagen-induced arthritis (CIA) model and the anti-collagen antibody induced arthritis (ACAIA) model, which show the above hallmarks of disease has allowed the identification of the cells and cytokines involved in the pathogenesis of the disease (5,6).

Many of the current therapies designed for RA focus on reducing the inflammation present within the joints but do not impact on the process of bone erosion; therefore one of the current goals in RA research is to inhibit the bone destruction that occurs (7). This exciting new field of research is known as osteoimmunology and it is beginning to highlight the link between the immune system and the skeletal system in the development, progression and establishment of RA (8).

The skeletal system consists of bone, cartilage and the connective tissues that connect the bones. Bone comprises of a solid matrix containing hydroxyapatite crystals, collagen fibres and cells. The main types of cells present within the bone are osteocytes, osteoblasts, osteoprogenitor cells and osteoclasts. Osteocytes are mature cells that maintain the bone matrix by dissolving and rebuilding it; osteoprogenitor cells are mesenchymal stem cells that differentiate into osteoblasts; osteoblasts are immature cells that produce new bone matrix, a process known as osteogenesis; and osteoclasts are multinucleated cells of the monocyte/macrophage lineage that degrade bone using hydrochloric acid and enzymes such as cathepsin K and matrix metalloproteinases in a process known as bone resorption (9). In a healthy individual there is a delicate balance between the number and function of osteoblasts and osteoclasts present within the joint, ensuring that in the normal process of bone remodeling the bone that is degraded is replaced. In an individual affected by RA several factors result in an increase in the number and function of osteoclasts, offsetting this balance and causing destructive bone erosion (10).

Interestingly, this field of osteoimmunology is beginning to pinpoint the inflammatory processes present within the arthritic joint, which are driving the osteoclast differentiation and activation. RA was previously thought to be a Th1 mediated disease; however, research has shown that it is most likely that Th17 cells are involved in the pathogenesis of RA, and it has now been established that there is a link between the Th17 cells which are found in the joint and osteoclast function (10,11).

IL-6 along with TGF-β in the presence of IL-23 induces Th17 cell differentiation. IL-6, IL-23 and TGF-β are all produced by macrophages; IL-23 is also produced by activated dendritic cells and TGF-β by synovial fibroblasts (1,3). Th17 cells produce several cytokines including IL-17A, IL-17F, IL-21 and IL-22 (8). IL-17A, which has been found in high concentrations in the synovium and synovial fluid of patients with RA, has multiple functions. It indirectly induces RANKL (receptor activator of NF- κB ligand) expression by synovial macrophages to produce IL-1 and TNF-α, and directly induces expression of RANKL on synovial fibroblasts and osteoblasts (12). RANKL binds to RANK on osteoclast precursor cells and allows these cells to differentiate into mature osteoclasts. Th17 cells also express RANKL, however the current literature shows that Th17 cells alone cannot induce osteoclastogenesis, osteoblasts are also required (11). This may be due to the fact that Th17 cells also produce a low amount of IFNγ, which is known to inhibit the differentiation of osteoclast precursor cells into mature osteoclasts (11).

Along with T cells, macrophages, neutrophils, mast cells and B cells are known t infiltrate the joint and contribute to the ongoing inflammation (1,4). Synovial macrophages express IL-1, IL-6 and TNF-α which are involved in the process of bone resorption (see Figure 1). IL-1 binds to the IL-1 receptors present on mature osteoclasts and TNF-α binds to TNF receptors present on osteoclast precursor cells. Both cytokine-receptor interactions trigger the expression of the transcription factor NF-κB, which allows the activation of osteoclasts and differentiation of precursor cells, respectively (12). TNF-α also induces the expression of RANKL on synovial fibroblasts and osteoblasts, and TNF receptors on osteoclast precursor cells, both of which are important in the differentiation and activation of osteoclasts (8,11,12).

Several interesting developments have already been made within this exciting new field of research. Lubberts et al showed that use of anti-IL-17A in the collagen induced murine model of RA decreased RANKL expression on synovial fibroblasts and osteoblasts and also decreased clinical arthritis scores observed (13); and Sato et al have shown that there is a positive correlation between IL-23 and RANKL expression in the synovium of patients with RA(11). Both studies therefore positively maintain the theory that Th17 cells represent a target for further therapeutic studies in RA. As this new cross-over field highlights further links between the bone remodeling process and the immune system, the prospect for new therapies which aim to tackle the inflammation and the bone erosion in RA looks promising.

References

McInnes, I.B. and Schett, G. Nature Reviews Immunology. 7, 429-442 (2007).
Birnbaum, H. et al. Current Medical Research and Opinion. 26(1), 77-90 (2010).
Brennan, F.M. and McInnes, I.B. The Journal of Clinical Investigation. 118(11), 3537-3545 (2008).
Cascão, R. et al. Neutrophils in rheumatoid arthritis: Autoimmunity Reviews. 9, 531-535 (2010).
Brand, D.D. et al. Springer Seminars in Immunopathology. 25, 3-18 (2003).
Nandakumar, K.S. and Holmdahl, R. Arthritis Research and Therapy. 8, 223 (2006).
van Vollenhoven, R.F. Nature Reviews Rheumatology. 5, 531-541 (2009).
Okamoto, K. and Takayanagi, H. International Immunopharmacology (2010). doi: 10.1016/j.intimp.2010.11.010
Martini, F.H. Fundamentals of Anatomy and Physiology, 7^th Edition.
Sato, K. Allergology International. 57, 109-114 (2008).
Sato, K. et al. The Journal of Experimental Medicine. 203 (12), 2673-2682 (2006).
Adamopoulos, I.E. and Bowman, E.P. Arthritis Research and Therapy. 10, 225 (2008).
Lubberts, E. et al. Arthritis and Rheumatism. 50(2), 650-659 (2004).

Anti-inflammatory cytokines as potential targets for neuropathic pain

MD Biosciences — Fri, 15 Apr 2011 14:29:00 GMT

We are continuing our series on the immune system, inflammation related factors and potential drup targets that fall in the overlap of the immune and nervous system. Our last discussion covered the pro-inflammatory cytokines and their relevance to neuropathic pain. This week we will cover anti-inflammatory cytokines.

IL-4

IL‐4 is released from activated mast cells and T cells and exerts anti-inflammatory effects by inhibiting release of IL‐1β, IL‐6, and TNF, promoting T cell differentiation into the anti‐inflammatory Th2 phenotype, and preventing macrophage and microglia activation. IL‐4 acts via two different heterodimers of IL‐4R. Using the spinal nerve ligation (SNL, Chung) model, pre‐treatment with IL‐4 delays onset of pain hypersensitivity and post‐treatment eliminates pain. These effects are associated with reductions in IL‐1β and microglial activation (1).

IL-10

IL‐10 is released from activated lymphocytes, macrophages, and mast cells and is a potent anti-inflammatory cytokine, known to inhibit the release of IL‐1β, IL‐6, and TNF. IL‐10 functions through binding its heterodimeric IL‐10Rα/β complex. In several preclinical neuropathic pain models, intrathecal administration of IL‐10 prevents or reverses pain hypersensitivity. IL‐10 is thought to act via inhibition of pro‐inflammatory cytokine release and thus reduction in recruitment and activation of other immune and immune‐like glial cells (1).

TGF‐β

TGF‐β is a pleiotropic, anti-inflammatory cytokine that acts via the TGF‐βRIII/RII heterodimeric complex. Intrathecal application of TGF‐β decreases development of pain hypersensitivity and eliminates existing pain in several preclinical models of neuropathic pain. These effects were associated with reductions in microglial proliferation, microglial and astrocytic activation, and neuronal expression of MCP‐1 (1).

Reference

Austin, PJ and Moalem-Taylor, G (2010) The neuro-immune balance in neuropathic pain: involvement of inflammatory immune cells, iimmune-like glial cells and cytokines. Journal of Neuroimmunology, 229 (1-2):26.