Gurwattan S. Miranpuri

The role of cation-dependent chloride transporters in neuropathic pain following spinal cord injury

BackgroundAltered Cl- homeostasis and GABAergic function are associated with nociceptive input hypersensitivity. This study investigated the role of two major intracellular Cl- regulatory proteins, Na+-K+-Cl- cotransporter 1 (NKCC1) and K+-Cl- cotransporter 2 (KCC2), in neuropathic pain following spinal cord injury (SCI).ResultsSprague-Dawley rats underwent a contusive SCI at T9 using the MASCIS impactor. The rats developed hyperalgesia between days 21 and 42 post-SCI. Thermal hyperalgesia (TH) was determined by a decrease in hindpaw thermal withdrawal latency time (WLT) between days 21 and 42 post-SCI. Rats with TH were then treated with either vehicle (saline containing 0.25% NaOH) or NKCC1 inhibitor bumetanide (BU, 30 mg/kg, i.p.) in vehicle. TH was then re-measured at 1 h post-injection. Administration of BU significantly increased the mean WLT in rats (p < 0.05). The group administered with the vehicle alone showed no anti-hyperalgesic effects. Moreover, an increase in NKCC1 protein expression occurred in the lesion epicenter of the spinal cord during day 2–14 post-SCI and peaked on day 14 post-SCI (p < 0.05). Concurrently, a down-regulation of KCC2 protein was detected during day 2–14 post-SCI. The rats with TH exhibited a sustained loss of KCC2 protein during post-SCI days 21–42. No significant changes of these proteins were detected in the rostral region of the spinal cord.ConclusionTaken together, expression of NKCC1 and KCC2 proteins was differentially altered following SCI. The anti-hyperalgesic effect of NKCC1 inhibition suggests that normal or elevated NKCC1 function and loss of KCC2 function play a role in the development and maintenance of SCI-induced neuropathic pain.

Role of NKCC1 and KCC2 in the development of chronic neuropathic pain following spinal cord injury

Neuropathic pain is a common problem following spinal cord injury (SCI). Effective analgesic therapy has been hampered by the lack of knowledge about the mechanisms underlying post‐SCI neuropathic pain. Current evidence suggests GABAergic spinal nociceptive processing is a critical functional node in this complex phenotype, representing a potential target for therapeutic intervention. Normal GABA neurotransmission is dependent on precise regulation of the level of intracellular chloride, which is determined by the coordinated activities of two cation/chloride cotransporters (CCCs) in the SLC12 family: the inwardly directed Na+‐K+‐Cl− cotransporter isoform 1 (NKCC1) and outwardly directed K+‐Cl− cotransporter isoform 2 (KCC2). Inhibition of NKCC1 with its potent antagonist bumetanide reduces pain behavior in rats following SCI. Moreover, the injured spinal cord tissues exhibit a significant transient upregulation of NKCC1 protein and a concurrent downregulation of KCC2 protein. Thus, imbalanced function of NKCC1 and KCC2 may contribute to the induction and maintenance of the chronic neuropathic pain following SCI.

HEMOCYTE POPULATION CHANGES DURING THE IMMUNE RESPONSE OF AEDES AEGYPTI TO INOCULATED MICROFILARIAE OF DIROFILARIA IMMITIS

Ultrastructural and lectin-binding studies have established that the melanotic encapsulation reaction of Aedes aegypti Liverpool strain against inoculated Dirofilaria immitis microfilariae (mff) is a hemocyte-mediated reaction. Total hemocyte counts from mff-inoculated (= immune-activated), saline-inoculated, and uninoculated female A. aegypti were determined using a hemocoel perfusion technique. Total hemocyte populations in uninoculated mosquitoes were significantly larger in younger mosquitoes, but no significant change was noted as mosquitoes aged beyond 14 days. Hemocyte populations in immune-activated mosquitoes increased from 1 to 3 days postinoculation (PI) and decreased on days 4 and 5 PI. Hemocyte populations at 1 to 4 days PI were significantly elevated in mff-inoculated A. aegypti as compared with saline-inoculated controls. Saline-inoculated mosquitoes displayed little change in total hemocyte numbers from 1 to 5 days PI, and their hemocyte populations were similar to those seen in uninoculated insects of the same age. Experiments involving the inoculation of [3H]thymidine along with mff or saline alone and studies involving the administration of colchicine suggest that increased hemocyte populations in immune-activated A. aegypti are a result of mitotic division of circulating blood cells.

B1 and TRPV-1 receptor genes and their relationship to hyperalgesia following spinal cord injury.

Study Design. Laboratory investigation of pain behavior following spinal cord injury. Objective. To explore changes in the spinal cord expression of nociceptive genes following spinal cord injury (SCI) as they relate to the manifestation of pain behavior in rats. Summary of Background Data. Neuropathic pain following SCI is common, disabling, and largely untreatable. In peripheral nerve injury models, bradykinin B1 and vanilloid 1 (TRPV-1) receptor activity is associated with neuropathic pain behavior. We sought to examine the role of these gene products in SCI-mediated pain. Methods. Rats were subjected to SCI using the MASCIS impactor. Animals were tested preinjury and at regular intervals postinjury for the appearance of thermal hyperalgesia using a hind limb withdrawal latency test. The expression of B1 and TRPV-1 genes was assessed using real-time polymerase chain reaction. Immunohistochemistry was used to localize the B1 and TRPV-1 receptors within the spinal cord. Results. Greater than twofold increases in the expression of the B1 and TRPV-1 genes were detected in the injured region of the spinal cord in animals exhibiting hyperalgesia compared with animals with SCI that did not display hyperalgesia. Immunohistochemical staining revealed that both receptor types were largely localized to the dorsal horn. Staining for TRPV-1 receptors decreased while that for B1 receptors increased in all of the injured animals when compared with sham-operated controls. Conclusion. B1 and TRPV-1 receptor genes are overexpressed in the injured spinal cord of animals manifesting thermal hyperalgesia following SCI compared with similarly injured animals without hyperalgesia. This finding is consistent with past work regarding the role of these receptors in nociception and indicates that ongoing modifiable processes are occurring in the spinal cord that lead to clinical pain syndromes.

Characterization of low-density lipoprotein uptake by murine macrophages exposed to Chlamydia pneumoniae

Exposure to Chlamydia pneumoniae is correlated with atherosclerosis in a variety of clinical and epidemiological studies, but how the organism may initiate and promote the disease is poorly understood. One pathogenic mechanism could involve modulation of macrophage function by C. pneumoniae. We recently demonstrated that C. pneumoniae induces macrophages to accumulate excess cholesterol and develop into foam cells, the hallmark of early atherosclerotic lesions. To determine if C. pneumoniae-induced foam cell formation involved increased uptake of low-density lipoprotein (LDL), the current study examined macrophage association of a fluorescent carbocyanine (DiI)-labeled LDL following infection. C. pneumoniae enhanced the association of DiI-LDL with macrophages in a dose-dependent manner with respect to both C. pneumoniae and DiI-LDL. Interestingly, increased association was inhibited by native LDL and occurred in the absence of oxidation byproducts and in the presence of antioxidants. However, enhanced DiI-LDL association occurred without the participation of the classical Apo B/E native LDL receptor, since C. pneumoniae increased DiI-LDL association and induced foam cell formation in macrophages isolated from LDL-receptor-deficient mice. Surprisingly, DiI-LDL association was inhibited not only by unlabeled native LDL but also by high-density lipoprotein, very low density lipoprotein, and oxidized LDL. These data indicate that exposure of macrophages to C. pneumoniae increases the uptake of LDL and foam cell formation by an LDL-receptor-independent mechanism.

Molecular evidence of repair and plasticity following spinal cord injury

Investigations into the genetic basis of neuronal damage following spinal cord injury have thus far been limited to the acute phase after the injury. Using microarray analysis, the present study compared the spinal-cord-injury-induced gene expression changes in adult rats at the epicenter and rostral segments of spinal cord at acute (12 h) and delayed (42 days) time points. We have previously reported that the acute response to spinal cord injury involves alterations in genes responsible for inflammation, cell cycle alteration, and altered receptor function. In contrast, the delayed response includes changes in the expression of HSP27, MAG, MAP-2, IGF-1 and ApoE. The alteration in expression of these genes suggests an ongoing repair process in animals whose functional recovery has reached a plateau.

Cannabinoid subtype-2 receptors modulate the antihyperalgesic effect of WIN 55,212-2 in rats with neuropathic spinal cord injury pain.

BACKGROUND CONTEXT There is increasing evidence for a role of the cannabinoid (CB) system in the development of neuropathic pain (NP) after spinal cord injury (SCI). The nonspecific CB₁ and CB₂ receptor agonists, WIN 55, 212-2 (WIN), have previously been shown to alleviate both mechanical and thermal hyperalgesia (TH) after peripheral nerve injury. PURPOSE The present study was designed to identify the CB receptors involved in the antihyperalgesic effect of WIN by using selective antagonists for CB₁ and CB₂ receptors. STUDY DESIGN This is an in vivo and behavioral study using a moderate T9 contusion SCI. After injury, TH of the hind paws was measured on postinjury days 21 through 42. METHODS Sprague-Dawley rats underwent a contusion SCI using the Multicenter Animal Spinal Cord Injury Study (MASCIS) weight-drop impactor, which induced a moderate T9 SCI. Only animals showing consistent plantar stepping and consistent forelimb and hind limb coordination (Basso, Beattie, and Bresnahan score=15) were tested for TH. Animals exhibiting decreased withdrawal latency time, indicating TH, on or before Day 42, were selected for pharmacological intervention. Animals not exhibiting TH did not receive pharmacological intervention and were sacrificed. Rats underwent hind paw testing before any drug administration (after injury), 45 minutes after selective CB antagonist (AM 251 or AM 630) administration (postantagonist) and again 45 minutes after WIN administration (post-WIN). There were a total of seven treatment groups: saline vehicle control; Dimethyl sulfoxide (DMSO) vehicle control; low-dose WIN (0.2 mg/kg); and high-dose WIN (2.0 mg/kg); AM 251 (3 mg/kg) and AM 630 (1 mg/kg) were given subcutaneously in a total volume of 0.5 mL. Followed by intraperitoneal injection of WIN after each antagonist, sham-operated rats repeated pharmacological intervention used with treatment Groups 5 and 6. RESULTS Thermal hyperalgesia was significantly ameliorated in a dose-dependent manner with systemically administered WIN. Cannabinoid receptor Type 1 antagonist AM 251 pretreatment did not affect the antihyperalgesic effect of WIN. By contrast, pretreatment with the CB₂ receptor antagonist AM 630 significantly attenuated the effect of WIN. CONCLUSION Taken together, these results suggest a role of the CB₂ receptor in modulating SCI-induced TH. Selective activation of the CB₂ receptor could potentially lead to analgesic effects on NP while avoiding psychotropic side effects in patients with SCI.

The Substitute Brain and the Potential of the Gel Model

This purpose of this paper is to review the recent history of the use of agarose gels. Although originally confined to electrophoresis work, agarose gels have proven themselves useful to a number of disciplines in the modern world, which includes brain infusion studies for research involving the treatment of various neurological conditions, such as Parkinson’s Disease. In reviewing the relevant research leading up to the modern day, this paper attempts to track agarose gels through their stages of accuracy verification, highlighting why they are useful to the neurosurgery discipline and characterizing the nature of their use. Agarose gels do have significant limitations, which are also discussed, but they have substantial potential as a modifiable medium or as a basis of comparison for even more accurate models in the future.

Neuropathic Pain: Role of Inflammation, Immune Response, and Ion Channel Activity in Central Injury Mechanisms

Neuropathic pain (NP) is a significant and disabling clinical problem with very few therapeutic treatment options available. A major priority is to identify the molecular mechanisms responsible for NP. Although many seemingly relevant pathways have been identified, more research is needed before effective clinical interventions can be produced. Initial insults to the nervous system, such as spinal cord injury (SCI), are often compounded by secondary mechanisms such as inflammation, the immune response, and the changing expression of receptors and ion channels. The consequences of these secondary effects myriad and compound those elicited by the primary injury. Chronic NP syndromes following SCI can greatly complicate the clinical treatment of the primary injury and result in high comorbidity. In this review, we will describe physiological outcomes associated with SCI along with some of the mechanisms known to contribute to chronic NP development.

Thermal hyperalgesia assessment for rats after spinal cord injury: developing a valid and useful pain index.

BACKGROUND CONTEXT Ongoing research to understand the mechanism behind pain is heavily dependent on animal testing. However, unlike humans, animal subjects cannot directly communicate with researchers to express the degree of pain they are experiencing. Therefore, measuring the presence of pain in animal studies is based on behavioral tests. The use of arbitrary values for determining the presence of pain in animal studies is an oversimplification of a complex and cortically dependent process. PURPOSE The purpose of the present study was to identify a statistically supported latency time indicator that can be used as an accurate index for hyperalgesia to thermal stimuli in Sprague-Dawley rats subjected to T9 contusive spinal cord injury (SCI). STUDY DESIGN A statistical analysis of latency of withdrawal from stimulus-mediated spinal reflex in 979 Sprague-Dawley rats that had been subjected to a T9 contusive SCI was performed. METHODS This is a retrospective review of a large research database derived from a series of studies performed evaluating thermal hyperalgesia in rats after SCI. Sprague-Dawley rats underwent a T9 contusive SCI and were tested for withdrawal latency from a heat stimulus. Assessment was done preinjury and on Postinjury Days 21, 28, 35, and 42 of the chronic phase of injury via a plantar withdrawal test. RESULTS The baseline test results of the 979 rats showed a significant resemblance to the normal distribution. The observed change in withdrawal showed mean latency drops of 0.42 second (standard error of the mean [SEM], 0.18; p=.026), 0.57 second (SEM, 0.19; p=.004), 0.63 second (SEM, 0.19; p=.002), and 0.69 second (SEM, 0.19; p=.0003). The standard deviation from the mean at all four postsurgical assessments was between 2.8 and 2.9 seconds. CONCLUSIONS Interpretation of withdrawal latency times as a marker for thermal hyperalgesia must be based on an appreciation for the normal distribution of pain scores. Recognizing that withdrawal latency is normally distributed both before and after injury allows for rational assignment of animals to groups designated as hyperalgesic and nonhyperalgesic. Two point nine seconds faster than the mean latency time is a statistically reliable indicator of thermal hyperalgesia in Sprague-Dawley rats subjected to contusive SCI. Repeated testing of animals to establish the presence or absence of thermal hyperalgesia beyond 21 days is not necessary in the absence of intervention.