Anti Allodynic Action Of The C5A Receptor Antagonist Biology Essay

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Diabetic Neuropathy (DN) defined as "the presence of signs or symptoms of peripheral nerve dysfunction in people with diabetes after excluding any other causes". Peripheral neuropathic pain is the most common complication of diabetes. The present study was designed to investigate the anti-allodynic effects of the C5a receptor antagonist, PMX-53, in a rat model of streptozotocin induced diabetic neuropathy. Following induction of diabetes, the rats developed a sustainable decrease in paw withdrawal threshold (PWT) response to the von Frey test within 5 weeks after a single intra-venous injection of streptozotocin (65mg/kg i.v.). The anti-allodynic effect of PMX-53 at 4 doses was examined at 5 weeks after induction of STZ. A siginificant increase in the PWT was observed following administration of PMX-53 sub-cutaneously. The alleviation of pain post-dose demonstrates the anti-allodynic and antinociceptive effect of PMX-53. Based on the experimental date, PMX-53 could become an efficacious therapeutic for the treatment of diabetic neuropathy.

1. Introduction

Peripheral neuropathic pain is a common complication of diabetes mellitus [1]. Up to 36 percent of patients with non-insulin dependent diabetes mellitus suffer from this condition which is believed to be progressive and irreversible [2]. The development of diabetic peripheral neuropathy is characterized by factors such as numbness, diminished sensation and pain. The pain associated with diabetic neuropathy is either simultaneous or due to exposure of mildly painful stimuli (hyperalgesia) or due to rather painful stimuli (allodynia) [3, 4]. Approximately 10 to 20 percent of patients suffering from diabetic peripheral neuropathic pain experience a burning, tingling or aching discomfort, pins and needles, shooting pain and hyperaesthesia have been reported [5]. It is also observed that the pain usually worsens at night disrupting the patient's sleep [6-8]. In other words, diabetic peripheral neuropathic pain interferes with the sleep quality, mood and activity level of patients. Another method of preventing the onset of the natural history of diabetic neuropathy can be the use of aerobic exercise training [9]. The initial management goal in the treatment of diabetic neuropathy is the control of hyperglycemia which may acutely worsen pain [10]. Among the different medications available for the treatment of diabetic neuropathy, only 30 to 50 percent reduction of pain is expected from patients on maximal dose of medication although complete relief is ideal [11, 12].

Diabetic neuropathies are in general a heterogeneous group of conditions involving different components of the somatic and autonomic nervous systems. It can be focal or diffuse, proximal or distal. Some of the causative factors include oxidative stress, microvascular insufficiency, nitrosative stress, defective neurotrophism, persistent hyperglycemia and autoimmune-mediated nerve destruction [13]. However, the mechanism, epidemiology and natural history of diabetic neuropathy are poorly understood. The reasons contributing to the poor understanding include the variable criteria for diagnosis, failure to recognize and diagnose the disease by many physicians as well as poor selection of patients for clinical trials and standardization of methodologies used for evaluation [14].

Diabetic neuropathy is defined as "the presence of signs or symptoms of peripheral nerve dysfunction in people with diabetes after excluding any other causes" [15]. Diabetic neuropathy has been considered as the most common and primary complication of long-term hyperglycemia [16, 17]. Studies have been performed to examine the mechanism underlying hyperglycemia induced nerve damage, but the pathogenesis of diabetic neuropathy remains unclear [18].

High blood glucose levels in patients suffering from diabetes mellitus for a long time causes an increase in the intake of glucose by endothelial cells lining the blood [19]. As a result of which, more glycoproteins are produced on their surface making the basement of the membrane thicker and weaker. Abnormality in the thickness of the vessels makes them weak, and therefore they start to bleed and reduce the flow of blood through the body [20]. In the case of diabetic neuropathy the lack of blood flow to the peripheral nerves is thought to play a role in the occurrence of neuropathy.

Another major complication caused by diabetes is the implication of the polyol pathway also called the sorbitol/aldose pathway that results in microvascular damage to nervous tissues and also to the retina and kidney [21]. The activation of the polyol pathway leads to the decreased levels of reduced NADP+ and oxidized NAD+, the most essential cofactors in redox reactions throughout the body. Due to high levels of glucose in people suffering from diabetes, any unused glucose will enter the polyol pathway and get converted to sorbitol. At normal levels of glucose, its affinity towards aldose reductase is less. On the other hand, at high levels of glucose, its affinity towards aldose reductase increases, meaning much higher levels of sorbitol and lower levels of NADPH, which gets used up as the pathway is activated. Excess activation of the pathway leads to high levels of extracellular and intracellular concentrations of sorbitol, increased concentrations of reactive oxygen species and reduced levels of nitric oxide and glutathione (production of both are promoted by NADPH). This imbalance causes damage to the cells as well as development of osmotic stress to the cell membranes [22]

However, these mechanisms do not provide complete relief as the management of diabetic neuropathic pain can be challenging for both patients as well as clinicians as the pain is only partially responsive or totally unresponsive to the existing pharmacological approaches [23, 24]. As a result of which there is a pressing need for the development of greater understanding related to the pathophysiology of neuropathic pain in patients with diabetes mellitus. In neuropathic pain associated with diabetes, a major role for peripheral nerves in the form of impulse generators has been proved. The sensory nerve fibers within the peripheral nerves and the distal terminal of their axons that are close to the peripherally innervated tissues as well as cell bodies of the dorsal root ganglion (DRG) that give rise to these peripheral axons, become hyperexcitable as a result of injury or disease. Improper spontaneity and

exaggerated responses to peripheral stimuli can be caused due to such hyperexcitability in both patients with diabetes as well as the animal models related to it [25 - 27]. The use of streptozotocin induced diabetic rat model has begun to shed lights in to the molecular processes that contribute towards hyperexcitability. These studies have proved that experimental diabetic neuropathy is associated with the expression of sodium channels which increases the excitability of DRG neurons [28, 29]. In the STZ rat model, in addition to the development of tactile allodynia, the levels of messenger RNA and proteins for the sodium channels Nav 1.3; 1.6; and 1.9 are found to be upregulated. This upregulation contribute to the electrogenesis in DRG neurons have been revealed by insitu hybridization and immunocytochemical studies [28]. These findings along with some recent studies have indicated that the hyperexcitability of and ectopic generation of impulses within the damaged peripheral sensory neurons and their axons strongly contribute to the pain associated with diabetic neuropathy.

The treatments for diabetic neuropathy are limited as the pathogenesis and aetiology of its symptoms are poorly understood. However, several classes of analgesic agents have been used for its treatment in patients suffering from painful diabetic neuropathy. The treatment for diabetic neuropathy is largely symptomatic comprising of analgesics, opiods, anticonvulsants, and tricyclic antidepressants [30]. The aetiological factors suggested include microvascular abnormalities, acute and chronic hyperglycemia, nitric oxide deficiency, and nerve compression, genetic and environmental variables [31]. Overall, the pathogenesis of diabetic neuropathy is multifactorial and the severity of neuropathy is determined by the pathological changes in the endoneural capillaries [32]. Diabetic neuropathy also shows resistance to non-steroidal anti-inflammatory drugs (NSAIDs) as well as to opioids such as codeine and morphine which in some cases do not provide pain relief [33]. Current first-line choices for the treatment for

diabetic neuropathy include antiepileptics, namely, pregabalin and gabapentin, and antidepressants, such as amitriptyline and duloxentine [34]. Another method of treatment that is prevalent is the use of sodium channel blockers. However, its clinical efficacy is a matter of controversy [35, 36]. Limited efficacy and low response to the current analgesic agents are the main drawback in the treatment of diabetic neuropathy despite the existence of various pharmacological approaches. This is mainly due to nature of diabetic neuropathy in which the severity of pain increases with the prolongation of the diabetic condition in patients. The efficacy of the analgesic agents could be affected by the changes in the etiological and pathophysiological mechanisms of pain development that takes place during the course of the disease [34]. As a result of the failure in the use of the efficacy of present analgesics, there is an alarming need for the development of new approaches in the treatment of diabetic neuropathy.

One of the possible candidates could be the use of the C5a antagonist for the treatment of diabetic neuropathy. The complement system is composed of over 30 proteins which are activated in the form of a cascade in response to injury and invading pathogens. C5a is a complement activation product which exhibits a major role in the genesis of inflammatory processes as well as in inflammatory diseases such as rheumatoid arthritis. It is one of the most important inflammatory mediators and its biological effects are mainly due to its binding with its G-protein coupled receptor, namely, C5aR1 present in various inflammatory cells such as neutrophils, monocytes and eosinophils. The role of C5a in neurological disorders is well-known as proved in rheumatoid arthritis as well as in neurodegenerative conditions such as Alzheimer's disease and Huntington's disease. Hence, it is believed that C5a could also play an important role in the development of pain in the case of diabetic neuropathy [35].

In the present study, we used a specific C5a receptor antagonist (PMX-53; AcF-[OP(D-Cha)WR]) to investigate its anti-alloynic effects in a rat model of streptozotocin induced diabetic neuropathy. PMX-53 belongs to class of selective small cyclic peptides and has demonstrated pseudo-irreversible and insurmountable antagonism to inflammatory cytokine release mediated through C5a in vitro. It has also demonstrated efficacy following intravenous, subcutaneous and oral administration in the treatment of several inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease. Hence, in the current study, the role of C5a receptor antagonist in the alleviation of pain in diabetic neuropathy was investigated.

Figure 1.2 Structure of PMX-53 [35]

Inducing experimental diabetes mellitus in a rat is the first step in the plan of testing the efficacy of the C5a receptor antagonist in the treatment of diabetic neuropathy. The streptozotocin (STZ) induced diabetic rat model is the most extensively studied model for diabetic neuropathy and its underlying mechanism. Streptozotocin is an antibiotic discovered from a strain of the soil microbe Streptomyces achromogenes and is diabetogenic due to a selective cytotoxic action on the pancreatic cells [36]. It is approved by the U.S. Food and Drug Administration (FDA) for the treatment of metastatic cancer of the pancreatic islet cells. STZ is a naturally occurring chemical that is particularly toxic to the beta cells in the islets of pancreas. Due to its risk of toxicity, it is only administered to patients whose cancer cannot be cured by surgery [37].

STZ is a glucosamine-nitrosourea compound and like any other alkylating agents of the nitrosourea class, it is also toxic to the cells as it causes damage to the DNA. STZ has a similar structure to glucose that allows its transport into cells by the glucose transport chain GLUT2, but it is not recognized by other glucose transporters. The beta cells in the islets high levels of GLUT2 may explain the selective toxicity of STZ in these cells [38, 39].

Figure1.3 Similarity in the structure of Streptozotocin and Glucose [38]

The method of evaluating the development of neuropathic pain following induction of diabetes and determination of efficacy of C5a receptor antagonist in alleviating pain is done by von Frey testing. Von Frey filaments are used to assess the pain threshold and response to putative treatments in STZ-induced diabetic rat model. It is the most widely used technique for the diagnosis of peripheral neuropathic pain. It is usually performed within the sciatic innervations of the hindpaw [40]. It was applied randomly to both the hindpaws to determine the intensity of stimulus required to elicit a paw withdrawal response.

Aims of the study

Induction of Diabetic Neuropathy using Streptozotocin in rats.

Investigate the anti-allodynic effects of PMX-53, a C5a receptor antagonist in alleviating pain in the STZ rat model of diabetic neuropathy.

Determine the peak-time for anti-allodynic response following administration of PMX-53 as a single bolus sub-cutaneous dose.

Isolate tissues (dorsal root ganglions and spinal cord) and process them for Immunohistological studies at a later date.

2. Materials and Methods

Male Sprague-Dawley rats sourced from UQBR-AIBN, The University of Queensland were housed at the TetraQ animal house facility located at Steele Building, The University of Queensland. The animals had free access to food and water and experienced a 12-12 hour light-dark cycle according to the animal ethics. The project is registered under the animal ethics code 366/09.

2.1. Streptozotocin (STZ) - animal model

The rats were anaesthetized using 50:50 ration of isofluorane and oxygen inhalation followed by administration of ~ 500µl of Benzylpenicillin behind the neck. Baseline blood glucose levels were calculated before induction of STZ by using blood glucose meter. Animals received a single intravenous injection of STZ at the dose of 65mg/kg of the body weight. STZ was injected using two methods, by intraveneous tail-vein injection and by intra-venous surgery to the jugular vein. The rats were then kept in warm condition and returned to individual cages and monitored post-STZ induction until full recovery was observed.

2.2. Von Frey Testing

Paw withdrawal thresholds (PWT) were determined using von Frey filaments in all the rats prior to surgery and every week post surgery to see the development of neuropathic pain. The PWT values of the hindpaws should decrease be >6g which indicates the development of neuropathic pain in the rodents. The starting force of the filament used was 6g. The von Frey filament was applied to the planar surface of the rodent hindpaw. The pressure was applied in such a way that the filament formed an S-bend. If a paw withdrawal response was induced by the application of 6g, then the 4g filament is used. If the force

of 6g does not induce any response, then we use the 8g filament until a paw withdrawal response was observed. The score for PWT was calculated on the basis of the least pressure filament to produce a paw withdrawal response. In the case of no response to any of the filaments, the rats are assigned a PWT score of 20g.

2.3 Efficacy Testing of PMX-53

2.3.1. Treatment groups

Following the development of neuropathic pain, the rats are tested with different doses of PMX-53. The doses used for testing are 0.1mg/kg, 0.3mg/kg, 1mg/kg and 3mg/kg. The vehicle used for testing is 5% glucose solution. The testing was carried out in 4 phases with a washout protocol of 3 days between each testing.

Each phase of testing includes two rats for each dose and a vehicle. The PWT scores for each dose were determined using von Frey filaments. The dose for the highest response and the time for peak response are determined from the four phases of testing.

The rats were then administered with 3mg/kg of PMX-53 and euthanized at peak response. These rat tissues were processed for immunohistological assessment to be conducted at a later date.

2.3.2. Dosage Preparation

The doses were prepared in 5% glucose solution (in milliQ H2O). The doses prepared were 0.1mg/ml, 0.3mg/ml, 1mg/ml and 3mg/ml of PMX-53. All the rats were weighed and the doses were calculated based on their weight (dosed at 1ml/kg). The doses were administered as a single bolus sub-cutaneous dose.

2.3.3. Protocol used for testing

The rats were weighed and allowed to acclimatize in the metabolic cages for 30 minutes. Then their baseline PWT score were determined thrice at an interval of 5 minutes and their average value was calculated. Then the rats received a single bolus subcutaneous dose of PMX-53 using a 5/8 gauge needle and 1ml syringes and placed back in their cages and PWT scores determined at intervals of 15min, 30min, 45min, 1hr, 1.5hr, 2hr and 3hr post-dose.

2.4 Immunohistochemistry

The operated STZ rats were euthanized and perfused with 4% paraformaldehyde solution on development of neuropathic pain post surgery. All the rats received a single bolus dose of PMX-53 (3mg/kg) and euthanized at 45min, the time of maximum response determined by previous experiments.

2.4.1. Tissue Preparation Preparation of 4% Paraformaldehyde

Fresh 4% PFA was prepared for perfusion. In order to make 1L of 4% PFA, about 600 ml of double distilled water was heated to 60-65 °C in a 1 L beaker in a fume cupboard. To this, 40g of PFA (ProSciTech, Aust.) was added and stirred for 5-10 minutes. Then few drops of 10M NaOH were added until the PFA is completely dissolved. The solution was then cooled to room temperature by placing it on ice. Then 100 mL of 1M PBS was added to the solution and the pH was adjusted to 7.2 - 7.4 by adding few drops of 1M HCl. This solution was filtered using a 0.45 µm pore size filter and stored at 4°C. The PFA has an expiry of 7 days after which it is discarded. Rat Perfusion Protocol

Three 60mL syringes were prepared with 4% PFA and stored at 4°C. The tubing used for perfusion was filled with 0.1M PBS and a 60mL syringe of 0.1MPBS were attached to it. Then the rats were administered a single bolus subcutaneous dose of ~ 0.5 ml Pentabarbitone. A lateral incision was and the thorax cavity was opened by cutting each side of the rib cage a 18 gauge needle was inserted to the left ventricle of the heart at its apex. The needle was then clamped and the right atrium was cut to release the circulating fluids. Then the animal was perfused with 60 -80 ml of 0.1M PBS followed by perfusion with approximately 200 mL of 4% PFA. Dissection of Dorsal Root Ganglion tissues

Following perfusion the lumbar region of the spinal cord was then exposed and the dorsal root ganglions (DRG) were carefully dissected using a dissecting microscope and micro-dissection scissors and forceps. The DRG dissected were L4 to L6 from the lumbar region of spinal cord. The DRG in this part of the spinal cord are large enough and easy to handle. The DRG were placed individually in separate wells in a 12 well culture plate containing 1.5 mL of 4% PFA. The plates were then stored at 4°C for 2 hours. Post- Processing of Tissues

After post-fixation of tissues for 2 hours in 4% PFA, the tissues were washed thrice with 0.1M PBS and then placed in 30% Sucrose in PBS solution. The tissues are stored in this solution for a maximum of 7 days at 4°C. Then the tissues were then transferred to a solution containing 1:1 ration of 30% Sucrose in PBS and OCT. The tissues can be stored in this solution for 8 days before embedding in to separate moulds. Embedding of tissues

The DRG of each rat are embedded in to separate moulds. The moulds used for embedding are of the dimensions 24mm * 24mm * 5mm. A thin line of superglue was applied on the base of the mould. Then the dorsal and ventral nerves of DRG were placed on the glue and aligned. The mould was marked in one end with a marker pen to identify the orientation of the DRG in the mould. The orientation used was L4 ipsi, L4cont, L5ipsi, L5cont, L6ipsi and L6cont from left to right of the mould (ipsi - ipsilateral - injured paw; cont - contralateral - uninjured paw). Then the mould was filled with OCT in such a way that no air bubbles are formed. Any air bubbles found near the DRG were carefully removed using tweezers. Then the mould became set when placed in an ethanol/dry ice bath. Then the frozen block was stored at -20°C until ready for cutting.

Figure 2.1 Orientation of DRG in embedding mould [18]

3. Results:

3.1 Induction of Diabetes Mellitus

The induction of diabetes mellitus in the STZ rats was measured using the blood glucose meter at day 10 post-surgery. The water intake measurement from day 7 to day 10 also indicated the development of diabetes in the STZ rats. At the end of day 10, the development of diabetes is confirmed if the blood glucose level is ≥15mMol/L and the water intake ≥100mL/day. All the rats which fail to attain this are euthanized at the end of day 10.

Figure 3.1 Mean (±SEM) of Water intake measurement in mL of 12 rats from day 1 to 10 following STZ induction intravenously.

From the graph, it is evident that rats 7198052, 7197932 and 7529293 did not develop diabetes at the end of day 10 post-STZ induction and hence had to be euthanized by an injection of 0.5ml pentabarbitone followed by cervical dislocation.

3.2 Induction of Tactile Allodynia:

The development of tactile allodynia in the STZ rats was monitored using von Frey testing at 0, 7, 14, 21, 28, and 35 days and once every week post-surgery till the paw withdrawal threshold becomes less than 6g. The testing demonstrated a continuous decrease in the PWT scores (Figure 3.1). The PWT scores of both the hindpaws decreased from ~12g to ~4g post-STZ induction over a duration of 5 weeks. The induction of diabetes and the development of allodynia post-surgery validated the STZ-model for the induction of neuropathic pain in rodents. The development of allodynia was observed between 4 and 5 weeks post-STZ induction. The decrease in the PWT less than 6g indicates the development of tactile allodynia.

Figure 3.2 Induction of Tactile Allodynia. The mean (±SEM) of Paw Withdrawal Threshold (PWT) of the hindpaws following STZ induction intravenously. The decrease in the PWT below 6g (below the dotted line) at von Frey testing indicates the development of allodynia following STZ induction.

3.3 Efficacy testing of PMX-53:

The ability of PMX-53 to alleviate tactile allodynia was assessed for three-hour following administration of PMX-53. The efficacy testing was carried out as per a wash-out protocol (refer to section 2.3.1). More specifically the same rodent was used for testing of the different doses of PMX-53. There was a 3 day untreated period between each testing. The doses used for testing were 0.1mg/kg, 0.3mg/kg, 1.0mg/kg and 3.0mg/kg of PMX-53. The vehicle used for testing was 5% glucose solution which is used for making up the different doses of PMX-53. Totally nine animals were used for testing the different doses. Animals that received vehicle were assessed on each testing day.

Initially PMX-53 was administered as a single-bolus sub-cutaneous dose of 1ml/kg (n=9). The dose of 3.0mg/kg showed a significant increase in the PWT indicating an anti-allodynic and antinociceptive effect of PMX-53. The PWT scores for 3 mg/kg almost matched the baseline PWT scores of the rats before surgery. The dose of 1 mg/kg showed the next significant anti-allodynic response. However, the doses 0.1mg/kg and 0.3mg/kg did not show much increase in the PWT scores.

D:\Project BIOC 7003\graph of all dose testing.jpg

Figure 3.3 Mean(±SEM) PWT of the hindpaw of rats administered a single subcutaneous bolus doses of 0.1mg/kg, 0.3mg/kg, 1.0mg/kg and 3.0mg/kg of PMX-53. PMX-53 at 3.0mg/kg showed a significant increase in the PWT of the hindpaws. The remaining three doses also showed an increase in PWT scores, however, the maximum response was observed for 3.0mg/kg of PMX-53.

3.3 Time of Peak-response for 3mg/kg of PMX-53.

The time for peak response for the single bolus sub-cutaneous dose of 3.0mg/kg of PMX-53 was found to be 60minutes post-dose. The duration of action of the drug is about 90 minutes post-dose after which it ceases at 120 minutes. The graph showing the peak-response time is as given below.

D:\Project BIOC 7003\peak response time.png

Figure 3.4 Determination of the peak response time (3.0mg/kg of PMX-53). The PWT scores showed a sharp increase at 60 minutes post-dose and come back to baseline values at the end of three-hour of testing.

The peak time for the anti-allodynic response for PMX-53 (3mg/kg) administered as a single bolus subcutaneous dose was used as the collection time for dorsal root ganglion and the spinal cord that were to be used for histochemical analysis of C5a location.

To investigate the possible role of C5a receptors, rats were euthanized at 60 minutes post-dose, perfused and DRG and spinal cord removed for processing. The tissues were then perfused from outside by placing it in 4% paraformaldehyde solution at 4°C followed by storing the tissues in 30% sucrose solution for 5days at 4 °C. After which, the tissues were transferred to a solution containing 50:50 ratio of 30% sucrose in PBS and OCT. The tissues were then freezed in plastic moulds and stored at -70 °C before using them for immunohistochemistry studies.

4. Discussion:

The induction of diabetes with streptozotocin is the most commonly used animal model of painful diabetic neuropathy. Complications of diabetes like hyperglycemia, polyurea, polyphagia, hypoinsulinemia, and polydipsia accompanied by weight loss were observed in adult rats within three days of induction of strepotozotocin [41]. The water intake measurement from day's 7 to 10 post-STZ induction and the blood glucose measurement confirmed the existence of irreversible diabetes mellitus in the male rats. This method is extensively used around the world because it is simple, inexpensive and available method. In the present study, the development of tactile allodynia in the STZ-induced model of diabetes was used to evaluate the anti-allodynic activity of PMX-53 between weeks 4 and 7 post-STZ induction. The development of allodynia occurred within 5 weeks of STZ administration.

Several critical parameters play an important role in the development of painful neuropathic pain following induction of diabetes in rats. Firstly, adult rats are preferred for the STZ model. This is mainly because the use of female rats is generally avoided as they can result in potentially confounding effects of the estrus cycle in various behavioral assays. Also, the use of young adult rats is preferred (180-220g) as they are found to develop a more consistent neuropathic pain syndromes [42]. In other words, the effect of rat strain, vendor as well as the vendor's breeding colony may have significant effects on the development of diabetic neuropathy. In the present study, Sprague-Dawley rats from the same vendor have been used. Another important factor that can influence the development of neuropathic pain is the animal's diet which has to be constant throughout an experiment [43]. In general, diets containing plan proteins such as soy, alfalfa, etc. as they contain phytoesterogens and hormone-like chemicals which can affect the appearance of neuropathic pain [44].

The housing of the rats also plays a significant role in developing symptoms of allodynia. It is generally advised that they are housed in cages with solid floors and soft bedding. Due to excessive drinking of water, they develop polyuria and as a result of which not more than one rat are housed in a single cage. It is also taken care that the bedding of the cages is changed on alternative days in order to prevent any kind of sickness or infections due to wet bedding [43].

The most common problem encountered in the use of STZ model is the failure to induce diabetic hyperglycemia. It is possible that not all the rats inducted with streptozotocin become hyperglycemic. In the present study, out of the 12 rats injected with streptozotocin, three rats did not develop diabetes at the end of day 10 when they had a blood glucose of <15mMol/L and a water intake of less than 100g. The failure to induce diabetes in all the rats injected with STZ presents a problem in technique. One of the possible reasons for this could be the improper storage of STZ powder or because of the STZ powder or STZ solution or STZ-filled syringes were exposed to room light for an extended period of time. STZ degrades when exposed to light for a longer period of time and after ~ 15 minutes in solution. The use of low dose of STZ means this protocol will induce only ~ 70 - 80% of the inject SD rats. The use of high dose of STZ can result in the induction of diabetes in higher number of animals as reported in the literature. However, the use of high dose results in severe complications in the animals and they have been described "sicky" as they exhibit significant loss of body weight and show signs of toxicity in liver and kidney. Hence, these animals become inappropriate for the study of neuropathic pain in diabetic neuropathy.

The development of neuropathic pain in the diabetic rats was observed during weeks 3 and 7 post-STZ induction. The evaluation of PMX-53, the C5a receptor antagonist for the alleviation of neuropathic was tested after the paw withdrawal threshold of rats reduced to ≤6g. Various studies have demonstrated the link between neuropathic pain pathology and complement activation suggesting complement antagonism could be a therapeutically effective strategy in the treatment of patients with neuropathic pain. The use of PMX-53 has been proved efficacious in the treatment of various inflammatory disease models such as inflammatory arthritis and bowel disease. The use of PMX-53 for the treatment of inflammatory disease is mainly attributed to the pro-inflammatory and immune-regulatory biological activities of the complement C5a. The biological actions induced by C5a are mainly mediated by interaction between the complement C5a and cell surface G-protein coupled C5a receptors (C5aRs) located on numerous cells of myeloid and non-myeloid origin [34].

Complement activation is an important part of the defense mechanism in host response to an injury or infection. However, overactivation or under-regulation of C5a plays an important role in the pathogenesis of numerous immune and inflammatory conditions. Experimental studies in the past have shown C5a as a pathogenic mediator in various acute and chronic conditions such as rheumatoid arthritis, inflammatory bowel disease and Alzheimer's disease. Hence, the use of PMX-53, the most widely characterized small molecule C5a receptor antagonist, has been proved efficacious in alleviating pain in various inflammatory models.

The complement system involves a cascading series of components or proteins that act as early defense components of the immune system. Complement anaphylotoxins such as C3a and C5a play as potential chemotactic agents that help in the mediation and activation of immune and inflammatory cells [45]. In the case of abnormal activation of the complement system, C3a and C5a induce the release of various inflammatory cytokines and mediators that assist in the development of the disease process [46-48]. The central role played by these anaphlyotoxins in the inflammatory and immune pathways is a matter of concern. Hence, in the present study, we utilized the active cyclic peptide C5aR antagonist (PMX-53) in a rat model of STZ-induced diabetic neuropathy. It was anticipated that PMX-53 would effective in alleviating the painful condition of diabetic neuropathy. Like in other inflammatory conditions, it was expected that the antagonism of C5a receptors from binding to the complement C5a thereby blocking its biological activity and alleviating pain.

The administration of PMX-53 was done as a single bolus sub-cutaneous does in the present study. All four PMX-53 doses administered to allodynic rodents produced clearly visible anti-allodynic improvements in von Frey behavioural testing in a dose-related manner. Despite this, only the 3mg/kg dose of PMX-53 achieved statistical significance; however, 1mg/kg approached significance. The peak anti-allodynic response time was found to be 60 minutes post-dose. However, the anti-allodynic effect ceases at 120 minutes post-dose.

These results confirm that the anti-allodynic effect of PMX-53 is primarily due to inhibition of C5a receptors. The use of C5a receptor antagonist blocks the formation of mediators resulting in the disease process, thereby alleviating the pain in diabetic neuropathy. However, the reason for ceasing of the anti-allodynic action in a short period of time is a point for concern. The most likely reason could be excretion of the compound from the body of the animal results in the anti-allodynic response to cease. Another reason could be the saturation of the target receptors in the rodent that could lead to the lack of allodynic improvement after a certain period of time post-dose. In order to achieve better results, the dose of PMX-53 can be increased and the testing can be carried out to measure any improvement in the duration of action.

5. Conclusions and future directions:

PMX-53 has been found effective in inducing an anti-allodynic response in the STZ-rat model for diabetic neuropathy. An increase in the PWT scores post-dosage indicates the effect of drug in relieving neuropathic pain. The dose that proved efficacious was 3.0mg/kg which provided maximum relief of mechanical allodynia where increase in the PWT scores of the hindpaws match the baseline scores prior to surgery. The remaining doses also produced allodynic improvement with 1mg/kg approaching significance. Despite this, the dose of 3mg/kg of PMX-53 was the one to achieve statistical significance. However, the ceasing of response at 90 minutes post-dose presents an interesting question about the mechanism of drug action as the drug has shown to be efficacious for a longer period of time in other models of neuropathic pain.

Further studies can be made by increasing the dose of PMX-53 in order to obtain complete and prolonged relief for allodynia. Also, studies can be made by changing the route of administration of the drug to the rodent. Oral administration of PMX-53 could be another way of testing its efficacy in inducing maximum anti-allodynic response. Knock-out studies can be another method of determining the target receptor of PMX-53. Hence, a C5a receptor knockout mice can be used followed by efficacy testing with PMX-53 can provide some interesting results on determining the target receptor for the drug. Immunohistorchemistry studies of the dorsal root ganglions and the spinal cord could provide some interesting insights about the localization of the C5a receptors and the mechanism of drug action. As the anti-allodynic response of the drug ceases in a short period of time, expression level studies of the different receptors using quantitative PCR studies with the fresh tissues of DRG's and spinal cord could provide some interesting results about the target of drug action. This study can also reveal the upregulation of C5a receptors in conditions of neuropathic pain.