Upregulation of brain-derived neurotrophic factor in the sensory pathway by selective motor nerve injury in adult rats

Selective motor nerve injury by lumbar 5 ventral root transection (L5 VRT) induces neuropathic pain, but the underlying mechanisms remain unknown. Previously, increased expression and secretion of brain-derived neurotrophic factor (BDNF) had been implicated in injury-induced neuropathic pain in the sensory system. In this study, as a step to examine potential roles of BDNF in L5 VRT-induced neuropathic pain, we investigated BDNF gene and protein expression in adult rats with L5 VRT. L5 VRT induced a dramatic upregulation of BDNF mRNA in intact sensory neurons in the ipsilateral L5 dorsal root ganglia (DRG), in non-neuronal cells in the ipsilateral sciatic nerve, and in motoneurons in the ipsilateral spinal cord. L5 VRT also induced de novo synthesis of BDNF mRNA in spinal dorsal horn neurons and in glial cells in the white matter of the ipsilateral spinal cord. Consistent with the mRNA expression pattern, BDNF protein was also mainly upregulated in all populations of sensory neurons in the ipsilateral L5 DRG and in spinal neurons and glia. Quantitative analysis by ELISA showed that the BDNF content in the DRG and sciatic nerve peaked on day 1 and remained elevated 14 days after L5 VRT. These results suggest that increased BDNF expression in intact primary sensory neurons and spinal cord may be an important factor in the induction of neuropathic pain without axotomy of sensory neurons.     

Major histocompatibility complex class II expression by activated microglia caudal to lesions of descending tracts in the human spinal cord is not associated with a T cell response

Lesion-induced microglial/macrophage responses were investigated in post-mortem human spinal cord tissue of 20 patients who had died at a range of survival times after spinal trauma or brain infarction. Caudal to the spinal cord injury or brain infarction, a strong increase in the number of activated microglial cells was observed within the denervated intermediate grey matter and ventral horn of patients who died shortly after the insult (4–14 days). These cells were positive for the leucocyte common antigen (LCA) and for the major histocompatibility complex class II antigen (MHC II), with only a small proportion staining for the CD68 antigen. After longer survival times (1–4 months), MHC II-immunoreactivity (MHC II-IR) was clearly reduced in the grey matter but abundant in the white matter, specifically within the degenerating corticospinal tract, co-localising with CD68. In this fibre tract, elevated MHC II-IR and CD68-IR were still detectable 1 year after trauma or stroke. It is likely that the subsequent expression of CD68 on MHC II-positive microglia reflects the conversion to a macrophage phenotype, when cells are phagocytosing degenerating presynaptic terminals in grey matter target regions at early survival times and removing axonal and myelin debris in descending tracts at later survival times. No T or B cell invasion or involvement of co-stimulatory B7 molecules (CD80 and CD86) was observed. It is possible that the up-regulation of MHC II on microglia that lack the expression of B7 molecules may be responsible for the prevention of a T cell response, thus protecting the spinal cord from secondary tissue damage. Received: 12 October 1999 / Revised: 31 January 2000 / Accepted: 8 February 2000     

Distinct types of microglial activation in white and grey matter of rat lumbosacral cord after mid-thoracic spinal transection

The inflammatory response has been characterized in the lumbosacral segments (L4-S1) of rats after spinal transection at T8. Immune cells were identified immunohistochemically using antibodies to complement type 3 receptor, CD11b (OX-42), the macrophage lysosomal antigen, CD68 (ED1), major histocompatibility complex class II (MHC II), and CD163 (ED2), a marker of perivascular cells. One week after cord transection, OX-42+ microglial density had nearly doubled. In the white matter, microglia became enlarged, often with retracted processes. In contrast, microglia in the grey matter remained ramified although nearly half of those lying medially contained clusters of ED1+ granules. After 8 weeks, ED1+ (+/-MHC II) macrophages were prominent in regions of Wallerian degeneration extending from dorsolateral to ventral funiculi. Microglial density remained raised in grey matter, particularly in the ventral horns of L4/5. Ramified microglia expressing MHC II+ (+/-ED1) extended from deep in the dorsal columns and around the central canal to the ventral columns. More ED2+ (+/-MHC II) perivascular and meningeal cells were recruited and expressed ED1. Thus, distinct from their conversion into macrophages in the white matter, the activation of ramified microglia after degeneration in the grey matter involves expression of ED1 without morphologic transformation.     

Neuropeptide Y and Galanin Binding Sites in Rat and Monkev Lumbar Dorsal Root Ganalia and Spinal Cord and Effect of Peripheral Axotomy

Using monoiodinated peptide YY (PYY) and galanin as radioligands, and neuropeptide Y (NPY) fragments, the distribution of NPY binding sites and its subtypes Y1 and Y2, and of galanin binding sites, was investigated in rat and monkey lumbar (L) 4 and L5 dorsal root ganglia (DRG) and spinal cord before and after a unilateral sciatic nerve cut, ligation or crush. Receptor autoradiography revealed that [¹²⁵I]PYY bound to some DRG neurons and a few nerve fibres in normal rat DRG, and most of these neurons were small. NPY binding sites were observed in laminae I–IV and X of the rat dorsal horn and in the lateral spinal nucleus, with the highest density in laminae 1–11. [¹²⁵I]NPY binding was most strongly attenuated by NPY_13–36, a Y2 agonist, and partially inhibited by [Leu³¹,Pro³⁴]NPY, a Y1 agonist, in both rat DRG and the dorsal horn of the spinal cord. These findings suggest that Y2 receptors are the main NPY receptors in rat DRG and dorsal horn, but also that Y1 receptors exist. After sciatic nerve cut, PYY binding markedly increased in nerve fibres and neurons in DRG, especially in large neuron profiles, and in laminae III-IV of the dorsal horn, as well as in nerve fibres in dorsal roots and the sciatic nerve. Incubation with NPY_13–36 completely abolished PYY binding, which was also reduced by [Leu,³¹ Pro³⁴] NPY. However, the increase in PYY binding seen in laminae I–IV of the ipsilateral dorsal horn after axotomy was not observed after coincubation with [Leu³¹, Pro³⁴] NPY. NPY binding sites were seen in a few neurons in monkey DRG and in laminae I-II, X and IX of the monkey spinal cord. The intensity of PYY binding in laminae I-II of the dorsal horn was decreased after axotomy. Galanin receptor binding sites were not observed in rat DRG, but were observed in the superficial dorsal horn of the spinal cord, mainly in laminae I-II. Axotomy had no effect on galanin binding in rat DRG and dorsal horn. However, galanin receptor binding was observed in many neurons in monkey L4 and L5 DRG and in laminae I–IV and X of monkey L4 and L5 spinal cord, with the highest intensity in laminae I-II. No marked effect of axotomy was observed on the distribution and intensity of galanin binding in monkey DRG or spinal cord. The present results indicate that after axotomy the synthesis of NPY receptors is increased in rat DRG neurons, especially in large neurons, and is transported to the laminae I–IV of the ipsilateral dorsal horn and into the sciatic nerve. No such up-regulation of the NPY receptor occurred in monkey DRG after axotomy. The Y2 receptor seems to be the main NPY receptor in DRG and the dorsal horn of the rat and monkey spinal cord, but Y1 receptors also exist. The increase in NPY binding sites in laminae I–IV of the dorsal horn after axotomy partly represents Y1 receptors. In contrast to the rat, galanin binding sites could be identified in monkey lumbar DRG. No effect of axotomy on the distribution of galanin binding sites in rat or monkey DRG and dorsal horn was detected, suggesting their presence on local dorsal horn neurons (or central afferents).     

Induction of microglial reaction and expression of nitric oxide synthase I in the nucleus dorsalis and red nucleus following lower thoracic spinal cord hemisection

In the present study, immunohistochemical stainings for OX-6, OX-42, nitric oxide synthase I and II as well as nitrotyrosine were used to investigate possible correlation among microglial reactivity, nitric oxide synthase upregulation, peroxynitrite involvement and neuronal death in the nucleus dorsalis and red nucleus following lower thoracic spinal cord hemisection. Significant neuronal loss was found in the ipsilateral nucleus dorsalis and contralateral red nucleus after cord hemisection. A distinctive microglial reaction for OX-42 could be observed from one to four weeks post axotomy in the ipsilateral nucleus dorsalis; by contrast, it was observed on both sides of the red nucleus from one to three weeks following cord hemisection. The activated microglial cells showed some degree of hypertrophy. From the microglial immunoreactivity as well as their appearance, it was speculated that microglial activation might be beneficial or protective to the axotomized neurons. In normal and sham-operated rats, neurons of the nucleus dorsalis were not nitric oxide synthase I reactive. Three weeks after cord hemisection, neurons in the ipsilateral nucleus dorsalis below the lesion showed strong immunoreactivity. Neurons in the red nucleus that normally displayed weak nitric oxide synthase I immunoreactivity showed an increase on both sides of the nucleus. These results suggested that nitric oxide synthase I expression in the nucleus dorsalis following axotomy was synthesized de novo and might act as a neurotoxic agent. However, the bilateral increase in expression of nitric oxide synthase I in the red nucleus after lower thoracic cord hemisection was due to up-regulation of the constitutive enzyme and might have some neuroprotective function. Our results also suggested that peroxynitrite played no or little role in the neurodegeneration in the nucleus dorsalis and red nucleus following axotomy.     

Activation of p38 MAP kinase in the rat dorsal root ganglia and spinal cord following peripheral inflammation and nerve injury

The intrathecal administration of p38 MAP kinase (p38) inhibitor has been shown to reduce hyperalgesia. In the present study, we investigated the activation of p38 in the rat dorsal root ganglion (DRG) and spinal cord following peripheral tissue inflammation and nerve injury immunohistochemically. Peripheral inflammation and chronic constriction injury (CCI) of the sciatic nerve induced a significant increase in the percentage of phosphorylated (P-) p38-immunoreactive (IR) neurons, primarily small sized ones in bilateral DRGs. In contrast, following axotomy, a significant decrease in the percentage of IR neurons was observed in ipsilateral DRGs. In addition, a marked increase was observed in the number of P-p38-IR microglia in the ipsilateral laminae I-IV and IX of the spinal cord following peripheral inflammation, CCI or axotomy. These findings suggest that p38 may play an important role in hyperalgesia and the activation of the spinal microglia.     

Experimental autoimmune neuritis induces differential microglia activation in the rat spinal cord

The reactive spatial and temporal activation pattern of parenchymal spinal cord microglia was analyzed in rat experimental autoimmune neuritis (EAN). We observed a differential activation of spinal cord microglial cells. A significant increase in ED1⁺ microglia predominantly located in the dorsal horn grey matter of lumbar and thoracic spinal cord levels was observed on Day 12. As revealed by morphological criteria and by staining with further activation markers [allograft inflammatory factor 1 (AIF-1), EMAPII, OX6, P2X₄R], reactive microglia did not reach a macrophage-like state of full activation. On Day 12, a significant proliferative response could be observed, affecting all spinal cord areas and including ED1⁺ microglial cells and a wide range of putative progenitor cells. Thus, in rat EAN, a reactive localized and distinct microglial activation correlating with a generalized proliferative response could be observed.     

Effects of dexamethasone on microglial activation in vivo: selective downregulation of major histocompatibility complex class II expression in regenerating facial nucleus

Following axotomy of the facial nerve microglial cells in the facial nucleus become activated, proliferate, and newly express class I and class II major histocompatibility complex (MHC) antigens. Dexamethasone treatment, starting 2 days prior to axotomy at 1 mg/kg/day, selectively inhibited axotomy-induced MHC class II expression on microglial and perivascular cells. In contrast, MHC class I expression was not significantly affected, nor was the expression of other microglial activation markers and the light microscopic morphology of activated microglia. A recently suggested inducer of MHC expression in rat nervous tissue, neuronal gamma interferon-like immunoreactive material, was also unaffected, as was glial fibrillary acidic protein immunoreactivity as a marker for concomitant astroglial activation. The differential effects of the drug suggest the presence of distinct regulatory pathways for different aspects of microglial activation. Inhibition of class II expression on activated microglia might be one mechanism how glucocorticoids act in the suppression of neuroinflammatory disease.     

Microglial reactions after subcutaneous formalin injection into the rat hind paw

Microglia in primary afferent projection territories are activated and proliferate after peripheral nerve injury. However, it is not known whether stimulation of peripheral nerves by noxious stimuli applied to their receptive fields activates microglial cells in the spinal cord. This study was designed to investigate the response of microglia in the lumbar spinal cord and in the brainstem to a tonic noxious stimulus. Thirty-two male Sprague-Dawley rats received subcutaneous injections of 5% formalin (50 microliter) into the plantar surface of the right hind paw, and 24 rats were injected with 50 microliter saline as a control. The lumbar spinal cord and brainstem were evaluated for immunoreactivity (IR) to complement receptor C3bi (monoclonal antibody OX-42) and major histocompatibility complex class II (monoclonal antibody OX-6) on postinjection hours 0, 2, 4 and 8 and days 1, 3, 7, 14 and 28. A qualitative and quantitative increase of OX-42-IR microglial cells were observed in the medial portion of the dorsal horn and in the gracile nucleus of the brainstem on the side ipsilateral to the formalin injection, starting on days 1-3 and peaking on day 7 postinjection. OX-6-positive cells were scattered both in gray and white matter, but no difference was detected between the two sides of the spinal cord or between formalin-injected and control animals. This is the first study that reports that subcutaneous injection of formalin into the rat's hind paw induces microglial activation in the spinal cord as well as in the brainstem. Although we have not determined whether these responses result from nociceptor activity, peripheral inflammation, or degeneration of primary afferents and/or central neurons, this method provides a simple, effective and stable animal model that will permit the future study of the mechanisms that contribute to microglial activation and its pathophysiological consequences.     

Increased uptake and transport of cholera toxin B-subunit in dorsal root ganglion neurons after peripheral axotomy: Possible implications for sensory sprouting

In the present study we show that, in contrast to the rat, injection of cholera toxin B-subunit (CTB) into the intact sciatic nerve of Macaca mulatta monkey gives rise to labelling of a sparse network of fibers in laminae I–II of spinal cord and of some mainly small dorsal root ganglion (DRG) neurons. Twenty days after sciatic nerve cut, the percentage of CTB-positive lumbar 5 (L5) DRG neuron profiles increased from 11% to 73% of all profiles. In the spinal cord, a marked increase in CTB labelling was seen in laminae I, II, and the dorsal part of lamina III. In the rat L5 DRGs, 18 days after sciatic nerve cut, the percentage of CTB- and CTB conjugated to horseradish peroxidase (HRP)-labelled neuron profiles increased from 45% to 81%, and from 54% to 87% of all neuron profiles, respectively. Cell size measurements in the rat showed that most of the CTB-positive neuron profiles were small in size after axotomy, whereas most were large in intact DRGs. In the rat spinal dorsal horn, a dense network of CTB-positive fibers covered the whole dorsal horn on the axotomized side, whereas CTB-labelled fibers were mainly seen in laminae III and deeper laminae on the contralateral side. A marked increase in CTB-positive fibers was also seen in the gracile nucleus. The present study shows that in both monkey and rat DRGs, a subpopulation of mainly small neurons acquires the capacity to take up CTB/CTB-HRP after axotomy, a capacity normally not associated with these DRG neurons. These neurons may transganglionically transport CTB and CTB-HRP. Thus, after peripheral axotomy, CTB and CTB-HRP are markers not only for large but also for small DRG neurons and, thus, possibly also for both myelinated and unmyelinated primary afferents in the spinal dorsal horn. These findings may lead to a reevaluation of the concept of sprouting, considered to take place in the dorsal horn after peripheral nerve injury. J. Comp. Neurol. 404:143–158, 1999. © 1999 Wiley-Liss, Inc.     

Dynamics of microglial activation in the spinal cord after cerebral infarction are revealed by expression of MHC class II antigen

Microglial reactivity associated with induction of MHC class II (HLA-DR) antigen is a sensitive indicator for pathological events in the CNS. To assess the response of glial cells after lesions of supraspinal descending tracts, HLA-DR, CD68 and GFAP were studied immunohistochemically on spinal cord tissue of 5 patients who died after unilateral infarction of the middle cerebral artery territory, and 5 control cases. In patients who died shortly after a stroke (4–14 days) increased HLA-DR-immunoreactivity (HLA-DR-IR) could be observed in the intermediate grey matter and in the ventral horn. The CD68-IR was much less intense. After longer survival times (5 weeks to 4 months), HLA-DR-IR in the grey matter was clearly lower than that observed in the spinal cord of short survival times, but very abundant in the dorsolateral funiculus, specifically within the corticospinal tract. In white matter areas, CD68-IR was almost identical to the HLA-DR-IR. Within the grey matter, CD68-IR was similar to the control tissue. A moderate increase of GFAP-positive astrocytes could be seen only in the grey matter after longer survival times. It seems probable, that the dynamics of HLA-DR-positive microglia reflect the early phagocytosis of presynaptic terminals by microglia in target regions of descending fibre tracts. In the white matter, the removal of degenerating axons by phagocytosing microglia expressing HLA-DR and CD68 antigens is a slower process which occurs over a period of months.     

Immune cell involvement in dorsal root ganglia and spinal cord after chronic constriction or transection of the rat sciatic nerve

Chronic constriction injury (CCI) of the sciatic nerve in rodents produces mechanical and thermal hyperalgesia and is a common model of neuropathic pain. Here we compare the inflammatory responses in L4/5 dorsal root ganglia (DRGs) and spinal segments after CCI with those after transection and ligation at the same site. Expression of ATF3 after one week implied that 75% of sensory and 100% of motor neurones had been axotomized after CCI. Macrophage invasion of DRGs and microglial and astrocytic activation in the spinal cord were qualitatively similar but quantitatively distinct between the lesions. The macrophage and glial reactions around neurone somata in DRGs and ventral horn were slightly greater after transection than CCI while, in the dorsal horn, microglial activation (using markers OX-42(for CD11b) and ED1(for CD68)) was greater after CCI. In DRGs, macrophages positive for OX-42(CD11b), CD4, MHC II and ED1(CD68) more frequently formed perineuronal rings beneath the glial sheath of ATF3+ medium to large neurone somata after CCI. There were more invading MHC II+ macrophages lacking OX-42(CD11b)/CD4/ED1(CD68) after transection. MHC I was expressed in DRGs and in spinal sciatic territories to a similar extent after both lesions. CD8+ T-lymphocytes aggregated to a greater extent both in DRGs and the dorsal horn after CCI, but in the ventral horn after transection. This occurred mainly by migration, additional T-cells being recruited only after CCI. Some of these were probably CD4+. It appears that inflammation of the peripheral nerve trunk after CCI triggers an adaptive immune response not seen after axotomy.     

Evidence for invasion of regenerated ventral root afferents into the spinal cord of the rat subjected to sciatic neurectomy during the neonatal period

Sectioning the sciatic nerve of experimental animals at the neonatal stage triggers growth of afferent fibers in the ventral root. The present study examined the possibility that the regenerating fiber terminals grow into the spinal cord. The sciatic nerve on one side was cut in neonatal rats. After the rats were fully grown, either an electrophysiological or a histochemical study was performed. The results of electrophysiological experiments showed that stimulation of certain loci in the L5 spinal cord evoked antidromic potentials in the L5 ventral root with a long latency. Various evidence suggests that the long latency potentials are due to activation of C fibers. These C-fiber potentials were on average bigger and were elicited from more numerous loci on the side ipsilateral to the sciatic nerve lesion than on the contralateral side. Furthermore, stimulation of the spinal cord of unoperated normal rats rarely evoked such potentials. For the histochemical study, horseradish peroxidase (HRP) was injected into the L5 spinal cord after cutting the L4-L6 dorsal roots. A lot more cells in the L5 dorsal root ganglion (DRG) on the side ipsilateral to the sciatic nerve lesion were labeled with HRP transported retrogradely through the L5 ventral root than on the contralateral side. Control experiments showed that few DRG cells are labeled with HRP in normal unoperated rats. The combined results of the electrophysiological and histochemical studies suggest invasion of ventral root afferents into the spinal cord, given enough postoperative time. It is not known whether or not these terminals make functional synaptic contacts in the spinal cord.     

Microglia, but not astrocytes, react to sciatic nerve injury in aging rats

Peripheral nerve axotomy activates microglia and astrocytes within regions of brainstem or spinal cord from which the nerve arises. The present study demonstrates that unilateral sciatic axotomy in rats 2 to 18 months of age results in differing responses with age between these two glial populations. By 4 days postaxotomy, both astrocytes and microglia become activated in 2-month-old rats, whereas only the microglial population shows evidence of activation in rats 8 to 18 months of age.     

Immunohistochemical localization of calretinin in the dorsal root ganglion and spinal cord of the rat

Calretinin (CR). a recently identified calcium-binding protein, is present in nervous tissue, including sensory pathways, where it may play an important role in regulation of cellular activity. Using immunocytochemistry, we examined the cellular localization of CR in dorsal root ganglia (DRG) and spinal cord of normal rats and after multiple unilateral dorsal root ganglionectomies. In DRG, CR-immunoreactive cell bodies and axons were a small subpopulation (10%) of medium- to large-sized neurons. In the spinal cord, CR-like immunoreactivity (LI) in neurons and fibers was found in all laminae except motoneurons. Dense fiber networks were also found in Clarke's column. The densest staining of both cell bodies and fibers was in the superficial laminae, especially lamina II, and in the lateral spinal and lateral cervical nuclei. CR-immunoreactive fibers were also observed in the fasciculi cuneatus and gracilis. Fasciculus gracilis exhibited the greatest number of labeled axons at the lumbosacral levels, but few labeled axons were found at the rostral thoracic and cervical levels. In contrast, the corticospinal tract at the base of the dorsal column was devoid of CR-immunoreactive fibers. Unilateral multiple lumbar ganglionectomies resulted in a loss of CR-LI in the dorsal columns ipsilateral to the surgery. In the spinal gray matter ipsilateral to the ganglionectomies, CR-LI was reduced in Clarke's column and slightly enhanced in the medial third of lamina II. Our observations demonstrate a unique distribution pattern of CR-LI compared to other calcium-binding proteins in the spinal cord, and suggest a role for CR in nociceptive and proprioceptive pathways.     

Nitric oxide synthase-like immunoreactivity in lumbar dorsal root ganglia and spinal cord of rat and monkey and effect of peripheral axotomy

With the immunofluorescence technique, nitric oxide synthase (NOS)-like immunoreactivity (LI) was found in a few medium-sized and small sensory neurons in lumbar (L) 4 and L5 dorsal root ganglia (DRG) of normal rat, and in most of these neurons, NOS-LI coexisted with calcitonin gene-related peptide and sometimes with substance P and galanin. NOS-immunoreactive nerve fibers, terminals and small neurons were also located in the dorsal horn of the segments 4 and 5 of the rat lumbar spinal cord with the highest density in inner lamina II. Many NOS-positive neurons and fibers were seen in the area around the central canal. A sparse network of NOS-immunoreactive nerve fibers was found in the ventral horn. After unilateral sciatic nerve cut in the rat, the number of NOS-positive neurons increased in the ipsilateral L4 and L5 DRGs, mainly in medium and small neurons, but also in some large neurons and very small neurons. NOS-LI could now also be seen in the ipsilateral dorsal roots, and in an increased number of fibers and terminals in both outer and inner lamina II of the ipsilateral dorsal horn. The number of NOS-immunoreactive neurons in lamina II of the ipsilateral dorsal horn was reduced. In the monkey L4 and L5 DRGs, many small neurons were NOS-immunoreactive, but only a few weakly stained nerve fibers and terminals were found in laminae I-IV of the dorsal horn at L4 and L5 lumbar levels. A few NOS-positive neurons were present in lamina X. The number of NOS-immunoreactive neurons was somewhat reduced in DRGs 14 days after peripheral axotomy, but no certain effect was seen in the dorsal horn. These results, together with earlier in situ hybridization studies, demonstrate that axotomy in rat induces a marked upregulation of NOS synthesis in primary sensory neurons, thus suggesting a role for NO in lesioned sensory neurons. In contrast, no such effect was recorded in monkey, perhaps indicating distinct species differences. © 1993 Wiley-Liss, Inc.     

Vasoactive intestinal polypeptide (VIP) increases in the spinal cord after peripheral axotomy of the sciatic nerve originate from primary afferent neurons

Following sciatic nerve axotomy, vasoactive intestinal polypeptide (VIP) immunoreactivity increases dramatically in the central terminal areas of the nerve whereas other primary afferent neuropeptides are depleted. The contribution of the peripheral nerve to VIP increases in the spinal cord was investigated by performing sciatic nerve section alone, dorsal rhizotomy of the lumbar roots, axotomy and rhizotomy in combination or section of other peripheral nerves terminating in the same segments as the sciatic nerve. VIP, and for comparison, substance P (SP), cholecystokinin (CCK), somatostatin (SOM), were localized in the lumbar spinal cord and corresponding sensory ganglia using unlabeled antibody immunohistochemistry. After sciatic nerve section, SP, CCK and SOM were depleted in the lumbar dorsal horn whereas VIP increased. After rhizotomy alone all neuropeptide staining including VIP was depleted; axotomy followed by rhizotomy prodiced the same result. Axotomy of other peripheral nerves terminating in the lumbar cord increased the area of neuropeptide depletion but correspondingly increased the area of VIP staining. A large proportion of small and medium diameter dorsal root ganglion cells were stained for VIP after nerve section or axotomy but not after rhizotomy alone. A radical change in neuropeptide metabolism of dorsal root ganglion cells occurs after peripheral axotomy, in the form of a maked increase in VIP synthesis. An intact dorsal root is necessary for increases in VIP in the spinal cord indicating the primary afferent origin of the response.     

Development of microglia in the chick embryo spinal cord: Implications in the regulation of motoneuronal survival and death

The role of microglia during normal development of the nervous system is still not well understood. In the present study, a chick embryo model was used to examine the development of microglia in the spinal cord and characterize their changes in response to naturally occurring and pathological death of motoneurons (MNs). The microglial response to MN axotomy and the effects of microglial activation on MN survival were also studied. We found that: 1) macrophages/microglial cells were present in the spinal cord at early developmental stages (E3) and that they were recruited after normal and induced MN apoptosis; 2) although many microglial cells were seen phagocytosing apoptotic bodies, a proportion of dying cells were devoid of engulfing microglia; 3) axotomy of mature MNs was accompanied by microglial activation in the absence of MN death; 4) excitotoxic (necrotic) MN death provoked a rapid and massive microglial recruitment with phagocytic activity; 5) lipopolysaccharide‐induced microglial activation in vivo resulted in the death of immature, but not mature, microglia; and 6) overactivation of microglia modulated the survival of mature MNs, either by killing them or by enhancing their vulnerability to die in response to a mild injury. Taken together, these observations indicate that normal microglia do not play an active role in triggering apoptosis of developing MNs. Rather, they act as phagocytes for the removal of dying cells during the process of programmed cell death. © 2009 Wiley‐Liss, Inc.     

Neurons and glial cells differentially express P2Y receptor mRNAs in the rat dorsal root ganglion and spinal cord

We examined the precise distribution of mRNAs for six cloned rat P2Y receptor subtypes, P2Y1, P2Y2, P2Y4, P2Y6, P2Y12, and P2Y14, in the dorsal root ganglion (DRG) and spinal cord by in situ hybridization histochemistry (ISHH) with 35S-labeled riboprobes. In the DRG, P2Y1 and P2Y2 mRNAs were expressed by 15% and 24% of all neurons, respectively. Although each receptor was evenly distributed between neurofilament-positive and -negative neurons, P2Y2 was rather selectively expressed by TrkA-positive neurons. Schwann cells expressed P2Y2 mRNA, and the nonneuronal cells around the DRG neurons, perhaps the satellite cells, expressed P2Y12 and P2Y14 mRNAs. No ISHH signals for P2Y4 or P2Y6 were seen in any cellular components of the DRG. In the spinal cord, P2Y1 and P2Y4 mRNAs were expressed by some of the dorsal horn neurons, whereas the motor neurons in the ventral horn had P2Y4 and P2Y6 mRNAs. In addition, astrocytes in the gray matter had P2Y1 mRNA, and the microglia throughout the spinal cord expressed P2Y12 mRNA. P2Y14 mRNA was weakly expressed by putative microglia. These findings should provide useful information in interpreting pharmacological and electrophysiological studies in this field given the lack of highly selective antagonists for each P2Y receptor subtype.