Preclinical Drug Discovery Blog

Microglial involvement in Neuropathic Pain: 5 activation pathways

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.

1. TLR4 (toll-like receptor family member 4)
2. P2X4 (purinociceptor 4)
3. INF-g and CB2
4. MCP-1
5. 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

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

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?

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.

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

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

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

1. Austin, P.J. and Moalem-Taylor, G (2010) Nociceptors: the sensors of the pain pathway. Journal of Clinical Investigation. 120(11):823
2. Smith, H.S. (2010) Activated microglia in nociception. Pain Physician. 13:295
3. Leung, L and Cahill, C.M. (2010) TNF-alpha and neuropathic pain - a review. Journal of Neuroinflammation. 7(1):27
4. Stein, C. et al. (2009) Peripheral mechanisms of pain and analgesia. Brain Research Reviews. 60(1):90
5. 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
6. 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?

 

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:

1. 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. 
2. 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.
3. 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.
4. 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.
5. 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.
6. 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.

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

1. Stewart SH, Mainous AG III, Gilbert G. J Am Board Fam Pract 2002;15:437-442.
2. Taylor, Marcia L. Southern Medical Journal 2004.
3. Mainous AG, Pearson WS. Fam Med 2003;35:112-118.
4. Galkina E, Kadl A, Sanders J, Varughese D, Sarembock IJ, Ley K.  J Exp Med. 2006; 203: 1273–1282.
5. Jongstra-Bilen J, Haidari M, Zhu SN, Chen M, Guha D, Cybulsky MI.  J Exp Med. 2006; 203: 2073–2083.
6. Owain R. Millington, James M. Brewer, Paul Garside and Pasquale Maffia.  Methods In Molecular Biology. 2010; 616: part 3 193-206.
7. http://en.wikipedia.org/wiki/Statin.
8. Jain MK, Ridker PM: Nat Rev Drug Discov, 2005; 4: 977-987.
9. Ridker PM, Danielson E, Fonseca FA, Genest J, Gotto AM, Jr., Kastelein JJ, Koenig W, Libby P, Lorenzatti AJ, MacFadyen JG, Nordestgaard BG, Shepherd J, Willerson JT, Glynn RJ: N Engl J Med, 2008; 359: 2195-2207.
10. Nissen SE, Tuzcu EM, Schoenhagen P, Crowe T, Sasiela WJ, Tsai J, Orazem J, Magorien RD, O'Shaughnessy C, Ganz P: N Engl J Med, 2005; 352: 29-38.

Inflammation & Pain processing: Relevant preclinical efficacy models

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

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

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