Jessica Naissant Senior seminar II paper Professor

Jessica Naissant
Senior seminar II paper
Professor: Dr. S. Pryor
Mentor: Dr. P. Cadet

Title: The role of glial cells and chemokines in neuropathic pain

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I. Abstract:
Neuroglia cells are all other cell types aside from the neuron found in the nervous system. Glial cells are known as the support cells of the nervous system, they play a very important role in maintaining homeostasis of neurons. The nervous system is generally separated into two parts, 1) the central nervous system, which includes the brain and spinal cord and 2) the peripheral nervous system, which includes everything else. Both systems have different cells that play major roles in neuropathic pain. Neuropathic pain is the sensation of chronic aches and shooting or burning pain that is the result of nerve damage or malfunction in the nervous system. There will be pain that arises as a direct consequence of a lesion or disease affecting the somatosensory system, causing a drastic change in nerve function at an injury site and the surrounding nerves. There seems to be no exact cause of neuropathic pain but researchers believe that some possible causes are chemotherapy, alcoholism, diabetes, HIV, M.S., and many other autoimmune diseases. Due to pain, microglial cells are known for producing immune cells that are important in nociceptive transmission. Research shows, that following nerve injury, an innate and adaptive immune response is inevitable regardless if there is an injury site. Immune cells that typically play a role in neuropathic pain are macrophages, astrocytes, microglia and many other immune cells. In order to understand neuropathic pain we must understand the mechanisms of neuropathic pain in the central and peripheral nervous system.
II. Introduction:
Mechanisms in the PNS, such as peripheral sensitization are considered to be ectopic firing of nerves due to injury. There are many causes of the spontaneous firing in neuropathic pain, largely due to an increase in sub-threshold fluctuations of nerve fibers and change in membrane potentials of the dorsal root. There is an upregulation and reorganization of the sodium and calcium channels, change in the expression of proteins, and the response of immune and glial cells which are affected by these channels. This eventually leads to sensitization, which is an increased responsiveness of neurons to normal stimulation. The partially damaged neurons ultimately shift the stimulus response of pain to the left, lowering the threshold of neuron response to pain. (Dworkin et al., 2010; Kalso et al., 2004). There are many changes that occur following injury, allodynia and hyperalgesia are two common disorders that occur as a result of nerve injury. Allodynia is pain due to a stimulus which does not normally provoke pain. On the other hand hyperalgesia is an increased response to a stimulus which is normally painful. Using animal models with nerve injuries, the role of glial cells and the neurochemical effects were able to be studied extensively. Observed were many secondary changes that began to take place throughout the entire nervous system, but more importantly why it has become such a major clinical problem. A very common change in neurochemistry is chronic pain, which is often described as inflammatory pain due to tissue inflammation. Many other symptoms attributed to neuropathic pain are anxiety, depression, and insomnia, just to name a few. (Dworkin et al., 2010; Kalso et al., 2004). Neuropathic pain is been used to define numerous pain disorders that are not related to each other by bodily injury nor do they share common causation. This makes it very difficult to specifically point to one common cause or injury. Researchers have deduced that neuropathic pain evolves as a direct outcome of involuntary nerve damage. Damage to the connective tissue that surrounds nerves is composed of nociceptors that affect the nerve and lead to neuropathic pain. Unlike neural plasticity, the spinal cord essentially changes and reorganizes. Neural plasticity creates neurochemical alterations of nerve fibers that ultimately affect the dorsal root ganglion and spinal cord; this can determine the sensitivity and reaction of subjects suffering from neuropathic pain and treatment methods. Following nerve injury sensitization is common among peripheral nerve injuries. Research has shown that this occurs at the dorsal root and abnormal discharges that take place due to C- fibers are released. It is known that neurons that are typically in the dorsal horn obtain feedback from nociceptive and non-nociceptive nerve fibers. C-fibers are well known to be high threshold nerve fibers, (nociceptive), while A-beta fibers are considered to be low threshold nerve fibers, (non-nociceptive). Non-nociceptive nerve fibers carry information from stimuli that is not usually painful, and they tend to create connections with C- fibers, which have degenerated, the non-nociceptive fibers, or A-beta fibers move upwards and sprout out towards the outer most layer of the dorsal horn. The dorsal horn usually receives information from C-fibers, or secondary neurons, which are usually transporting pain signals to direct a pain response, therefore, setting a chain reaction of events that usually includes the release of immune cells and glial cells.
III. Discussion:
There are many different mechanisms involved in neuropathic pain that attribute to the expression of symptoms such as hyperalgesia and allodynia. Mechanisms in the CNS, such as spinal cord hyper-excitability, which is known as central sensitization, are changes that occur centrally in the spinal cord and dorsal horn. There is an increased in transport of signals that affect the sub threshold limit. There are many causes of spinal cord hyper excitability, a well-known cause is the malfunction of the NMDA receptor and the decrease of GABA; both play a role in the persistence of neuropathic pain.
Recent studies reveal how dysfunctions within the nervous system lead to neuro-inflammation and immune responses that in turn contribute to neuropathic pain. Nerve damage can initiate immune responses by the use of cytokines and chemokines. Chemokines and glial cells play a major role in sustaining homeostasis of nerves and neuronal development. The central nervous system plays a major role in the immune system because of the immune factors, which are the nervous systems response to certain harmful and potentially harmful stimuli. Signals travel to the spinal cord and the brain, where many different responses, physical and physiological, which ultimately create the experience of pain in sentient beings. Neuropathic pain is considered by many scientists to be a “neuro-immune disorder”, because it has been discovered that the activation of the immune and glial cells in the dorsal root ganglia and spinal cord results in pro and anti-inflammatory cytokines and chemokines as discussed throughout this paper.
Cytokines are small secreted proteins released by cells that have a specific effect on the interactions and communication between cells. They aid in cell to cell communication during an immune response and stimulate the movement of cells towards in jury sites, inflammation and infection. Chemokines, on the other hand, are considered to be a sub unit of cytokines. They are also tiny protein particles that form a larger unit of cell signaling particles known as cytokines. The purpose of both these molecules is to secrete movement from an organism (cell) in response to a chemical stimulus; this is known as chemotaxis, and takes place between adjacent cells. When cells deviate from their usual movement in response to the presence of chemical stimuli, cytokines and their sub particle counterparts, chemokines are in play. The presence of inflammation or injury triggers a release of chemicals that show immune cells where to travel to aid at the site of injury or inflammation. During this process neutrophils are stimulated to leave blood vessels and travel towards the injury site. Monocytes and other immune cell responses are engaged at a later stage. Chemokines are believed to play two major roles in neuropathic pain; the first is a responder role to chemical stimuli, therefore being considered pro-inflammatory. The other role is one of a homeostatic nature, due to the control of cell migration for normal tissue development and maintenance. Only 19 types of chemokine receptors have been identified in mammals so far. The chemokine family is divided into two major groups, CC and CXC, which are further divided into subgroups based on the type of stimulus and the tissue involved.
In this paper, a major focus will be the two studies done on C57Bl/6L mice, which had partial nerve damage to their sciatic nerve. Initially, observations were done by testing the pain threshold on injured mice by using behavioral test. Also, the immune response of neutrophils, dendritic cells, T cells, B cells, and macrophages via observation using immunohistochemistry. In the third study, MS, which is a chronic inflammatory disease is modeled in mice, known as the EAE model. The brain behavior and release of chemokines following nerve injury are studied in these mice.
IV. Purpose and hypothesis:
Cytokines and chemokines modulate the pain response by impacting both the immune and the nervous system cells. Spinal glial cells enhance and maintain neuropathic pain by releasing potent neuromodulators, such as pro-inflammatory cytokines and chemokines. Microglial cells are capable of synthesizing and secreting numerous chemicals that initiate immune responses. Chemical signals play an important role in nociceptive transmission in neuropathic pain. The development of neuropathic pain is associated with changes in levels of multiple complement system proteins. There must be a new therapeutic route not only in neurodegenerative diseases, but in neuropathic pain that can be found through the study of immune factors such as glial cells, cytokines and chemokines.
V. Method:
The mythology used in study 1 consisted of five groups of mice that was included in the study, four underwent sciatic nerve injury and a group of naïve rats that were not exposed to injury served as the control, ( Hanani et al., 2002; Jasmin et al., 2010; Ohara et al., 2008). Experiments were carried out on male C57BL/6J inbred mice (7-8 weeks old). The nerve models included partial sciatic ligation of the left thigh. The sciatic nerve was exposed proximal to the thigh and freed of adhering tissue. A partial ligation of the sciatic nerve was made by 9-0 silk. Sham-operated mice were used as the control group; the left sciatic nerve was exposed, but not ligated. Behavioral tests were done before partial ligation of the sciatic nerve and recorded after ligation and for 14 days after. Mechanical allodynia and hyperalgesia was assessed by placing mice in enclosures and stimulating the hind-paws using the tip of an electronic von Frey anesthesiometer (electro-rod) until the mice were able to move their paws in response to the stimulus. Immunohistochemistry was used to observe the response of immune cells after ligation. Following peripheral nerve injury in study one, mice that had symptoms of allodynia and hyperalgesia were observed. The behavioral responses are seen in Figure 1.
VI. Results:

Figure 1 is a depiction of C57BL/6J mice, which were injured via the partial sciatic nerve. Group A showed symptoms of mechanical allodynia while group B showed symptoms of thermal hyperalgesia, measurements of these injuries were studied and observed and discovered that both groups suffered from neuropathic pain symptoms, there was hypersensitivity in the hind-paw of the mice in groups A and B. Unlike the injured mice the uninjured mice showed little to no decrease in the hind-paws in both groups, as shown above. Prior to ligation paw withdrawal was measured in both groups of mice and thee results were that partial sciatic nerve ligation had a severe decrease of paw withdrawal threshold when stimulated. Just 2 days after ligation, injured mice in both groups showed a drastic decrease in paw withdrawal. This decrease remained consistent for the entire 2 week period that the experiment took place. All mice exhibited neuropathic pain symptoms, even those who were uninjured, but those who suffered injury showed a drastic decrease in paw withdrawal when stimulated.
Neuro-inflammation in the sciatic nerve was observed in Figure 2, unlike figure one, this study observed the immunoreactivity of cells 14 days post ligation and compared them to a control of mice that were uninjured but showed symptoms of neuropathic pain. Observed are the inflammatory changes in the sciatic nerve of immune cells such as neutrophils, macrophages, dendritic cells, T cells, and B cells, which were the 5 group A-E respectively. Using immunohistochemistry, the immune-reactivity of the varied immune cells is shown by the green color below.

Figure 2, demonstrates the immunoreactivity after ligation in immune cells. Observes are sciatic nerve injuries distal and proximal to injury site, and compared to sham operated mice, which represented the control group since there did not suffer injury. Group A showed the immunoreactivity of neutrophils and the results were that neutrophils were significantly higher in injured mice compared to the control group. On the first day nerves distal to the injury site were significantly more inflamed compared to the control group also. Nerves distal to the injury site also showed an increase in neutrophils compared to the proximal nerves. Group B, Macrophages, was also very high compared to the control group. Between days 1 and 14 we can see a consistent increase in macrophages in the injured mice. When comparing the image to neutrophils, the green hue from the immunohistochemistry test is significantly brighter and more abundant than that of neutrophils. Also the nerves proximal and distal to the injury site in group B were significantly higher than the neutrophils in group A. Group C, dendritic cells were higher in the injured mice but, the nerves distal and proximal to the injury site did not show an abundance of dendritic cells until days 5-14. Also the green hue is significantly less compared to groups A and B. Group D, T-cells, show a consistent increase of T-cells from day 1-8 after surgery, and then a decrease in T-cells between days 9-14. Group E, B-cells, was higher in injured mice but significantly lower compared to the responses of all other immune cells after surgery. However, there was a consistent increase in B-cells between days 3-14. The diagrams on the right side of each histogram show representative immune chemical responses post ligation. The number of immune reactive cells was determined in sciatic nerve injured mice in study 1. Next the observed studies were of the immune reactivity in the dorsal root ganglia.
In this study similar methodology was used, the use of C57BL/6J mice, partial ligations to the sciatic nerve, the only difference was the density of immune reactive cells at the L3-L5 DRG 14 days post ligation and compared them to the uninjured mice. Observed are the inflammatory changes in the dorsal root ganglia by noting the response of immune cells such as neutrophils, macrophages, dendritic cells, T cells, and B cells, which were the 5 groups, A-E respectively. The immune response at several different times post-surgery is an effective model of neuropathic pain. The configuration of neuro-inflammation post-surgery is used to identify the roles of glial cells but also the effectiveness of immune response after ligation. (Bailey and Ribeiro-da-Silva, 2006; Munglani et al., 1995).

In group A very few neutrophils were observed in the dorsal root ganglia compared to the control group. There was a slight increase between days 1-7 but there was not a significant difference until days 7-14. Group B, macrophages showed a significant increase between days 3-7 and began to decrease between days 8-14. Group C, dendritic cells were detected in very small amounts. Compared to the control (sham) group, days 3-14 showed a significantly higher amount of dendritic cells. Group D, T-cells, showed no significant difference between the control group and the injured mice until days 3- 14, T-cells consistently increased. Lastly group E, B cells, showed a slightly higher amount of B-cells compared to the sham operated group.
Finally, in paper two, scientist show how inflammatory mediators better known as cytokines and chemokines, are released from the injured nerve fibers and immune cells of mice. It is known that the activation of these inflammatory mediators is due to nerve injury, but using the EAE model, observations of mice experiencing symptoms of the auto immune disorder M.S. gives a deeper look into how neuropathic pain plays the role of anti-inflammatory and pro-inflammatory mediator after ligation. Chemokines have been shown to directly contribute to nociception by activating spinal glial cells which leads to pain hypersensitivity. This study observes how immune factor and mediators are already activated prior to injury. Unlike the studies shown before, chemokines CCL2 and CX3CR1 are involved in pain regulation and consistency in figure 4.

This model shows the brain behavior of mice following nerve injury in the EAE model. Following neuro-inflammation glial cells and other immune cells become reactive and increase the expression of chemokines and their respective receptors. Spinal nerve ligation prompts the increase of CX3CR1 in glial cells. Group A shows the reactivity of CX3CR1 in the spinal cord post ligation. Groups B-D show a double staining of CX3CRI co-localization in the dorsal horn, there is a significant reduction of membrane bound receptors. Also the similar patterns in these groups show how the influence of CXC3L1 alters the signals between glial cells and neurons. The cleaving of CXC3L1 increases the activity of microglia indication that neuropathic pain development may be a direct result of cleavage from the chemokine and glial cell interactions.
Figure 5 depicts the increased expression of CCL2 post ligation, groups A and B show a very similar patterning of chemokine behavior but group B has an increased release of CCL2 compared to A which has no injury site. Groups C and D show how CCL2 is constricted when observed in Gfap and NeuN. CCL2 drastically increases monocytes to sites of injury and inflammation. In mouse tissue it was seen by scientist that CCL2 binded with CCR2 (receptor) 10X higher than other chemoreceptors because of the receptors innate respone due to glial and immune cell interactions.
VII. Conclusion:

There have been many different ideas and tactics to deal with neuropathic pain, studies have been done on direct targeting of chemokine, stabilizing antibodies and immune cell responses. . Figure 6, shows the movement of nerve injury and the response of known chemokines. Synthesizing chemokines with known inhibitors can aid in cleavage patterns that can improve the symptoms of neuropathic pain. Overall management of neuropathic pain has been a challenge for a very long time. Research shows that chemokines role in regulating pain is extremely vital. Neuronal-glial interactions have a profound impact in clinical studies and further research by pharmaceutical companies can help to create a potent chemokine antagonist that aids in the chronic aches of neuropathic pain. All mice exhibited neuropathic pain symptoms showing the development of mechanical and thermal pain hypersensitivity of the hind-paw in study 1. Mechanical allodynia was observed for more than 2 weeks after the nerve injury and thermal hyperalgesia was observed for more than 2 weeks after injury. In the nerve samples from sham-operated mice, little to no immune cell factors was detected. After PSNL, a significant increase in the numbers of immune cells mostly neutrophils were observed at the site of injury at all-time points. Infiltration and activation of inflammatory cells differed between the peripheral and central nervous system, with similar cell types (e.g., neutrophils, dendritic cells, T cells, and B cells) observed in the injured sciatic nerve and DRGs, but not in the spinal dorsal and ventral horns, following PSNL. In study 2 the data indicated that neutrophils, macrophages, dendritic cells and lymphocytes do not infiltrate the lumbar spinal cord following PSNL in mice. Inflammatory changes involving activation of microglia and astrocytes in the spinal cord after peripheral nerve injury, which contributes to hypersensitivity and chronic neuropathic pain. Following CNS inflammation, microglia and astrocytes become reactive and increase the expression of chemokines and chemokine receptors
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