Distribution and expression of tissue inhibitors of metalloproteinase in dorsal root entry zone and dorsal column after dorsal root injury

To understand whether tissue inhibitors of metalloproteinase (TIMPs) contribute to the failure of regenerating sensory axons to enter the spinal cord, we used in situ hybridization and immunocytochemistry to examine the expression of TIMP1, TIMP2, and TIMP3 in the dorsal root, dorsal root entry zone (DREZ), and dorsal column after dorsal root injury in adult rats. We found that the three TIMPs and their mRNAs were up-regulated in a time-, region-, and cell-type-specific manner. Strong up-regulation of all three TIMPs was seen in the injured dorsal roots. TIMP2 was also significantly up-regulated in the DREZ and degenerating dorsal column, where TIMP1 and TIMP3 showed only moderate up-regulation. Most cells up-regulating the TIMPs in the DREZ and degenerating dorsal column were reactive astrocytes, but TIMP2 was also up-regulated by microglia/macrophages, especially at long postoperative survival times. These results suggest that TIMPs may be involved in controlling tissue remodelling following dorsal root injury and that manipulation of the expression of TIMPs may provide a means of promoting axonal regeneration into and within the injured spinal cord.     

Phosphorylation of CREB in thoracolumbar spinal neurons and dorsal root ganglia after renal artery occlusion in rat.

These studies have demonstrated that ipsilateral renal artery occlusion (RAO) in rat results in the phosphorylation of cyclic AMP (cAMP) response element binding protein (p-CREB) in the thoracolumbar (T8-L2) spinal cord and associated dorsal root ganglia (DRG). p-CREB-immunoreactivity (IR) was expressed bilaterally in the thoracolumbar spinal cord, whereas expression in the DRG was ipsilateral relative to RAO. p-CREB-IR was primarily expressed in four distinct regions of the spinal cord: medial or lateral dorsal horn (MDH or LDH), dorsal commissural nucleus (DCN) and the region of the intermediolateral cell column (IML). After RAO, p-CREB-IR was greatest in the T13-L2 spinal segments. Within the T13-L1 spinal segments, p-CREB-IR was greatest in the MDH, LDH and DCN and expression in each of these regions was comparable within a segment. Following RAO, there was a significant (p < or = 0.001) increase in the percentage (86-98%) of p-CREB-IR spinal neurons expressing choline acetyltransferase (ChAT)-IR (a marker of preganglionic neurons) in the IML of the T10, T12 and L1 spinal segments examined. After ipsilateral RAO, expression of p-CREB-IR was increased in the ipsilateral, T8-L2 DRG with the greatest number of p-CREB-IR dorsal root ganglion cells being located in the L1 dorsal root ganglion. Retrograde tracing with Fluorogold (FG) to label renal afferent cells in the DRG revealed a significant (p < or = 0.01) increase in the percentage (75-86%) of renal afferent cells expressing p-CREB-IR after ipsilateral RAO. These studies demonstrate that p-CREB-IR is a useful tool for examining the distribution of spinal neurons and DRG involved in reflexes of renal origin. In addition, expression of p-CREB-IR may be coupled to late response genes that may exert long-term changes in neuronal function after RAO.     

NGF mRNA is expressed in the dorsal root ganglia after spinal cord injury in the rat

Nerve growth factor (NGF) mRNA is expressed in a variety of cell types in the injured spinal cord and its protein implicated in both positive and negative neurological outcomes of cord injury. Here we demonstrate that NGF mRNA is also upregulated in dorsal root ganglion (DRG) neurons after spinal cord injury and that the percentage of sensory neurons expressing NGF mRNA correlates with proximity to the lesion epicenter. Our data suggest that, in DRG, NGF gene expression may be upregulated by damage to the central processes of sensory neurons.     

Time course of substance P expression in dorsal root ganglia following complete spinal nerve transection

Recent evidence suggests that substance P (SP) is up-regulated in primary sensory neurons following axotomy and that this change occurs in larger neurons that do not usually produce SP. If this is so, then the up-regulation may allow normally neighboring, uninjured, and nonnociceptive dorsal root ganglion (DRG) neurons to become effective in activating pain pathways. By using immunohistochemistry, we performed a unilateral L5 spinal nerve transection on male Wistar rats and measured SP expression in ipsilateral L4 and L5 DRGs and contralateral L5 DRGs at 1-14 days postoperatively (dpo) and in control and sham-operated rats. In normal and sham-operated DRGs, SP was detectable almost exclusively in small neurons (< or =800 microm2). After surgery, the mean size of SP-positive neurons from the axotomized L5 ganglia was greater at 2, 4, 7, and 14 dpo. Among large neurons (>800 microm2) from the axotomized L5, the percentage of SP-positive neurons increased at 2, 4, 7, and 14 dpo. Among small neurons from the axotomized L5, the percentage of SP-positive neurons was increased at 1 and 3 dpo but was decreased at 7 and 14 dpo. Thus, SP expression is affected by axonal damage, and the time course of the expression is different between large and small DRG neurons. These data support a role for SP-producing, large DRG neurons in persistent sensory changes resulting from nerve injury.     

NGF levels decrease in the spinal cord and dorsal root ganglion after spinal hemisection

To examine changes in nerve growth factor (NGF) levels in spinal cord and dorsal root ganglia (DRG) after spinal injury, male Sprague-Dawley rats weighing 150-175 g were given spinal hemisections. NGF content was measured at various post-surgical times and compared with naive controls (n = 4 per time point) in the spinal cord, DRG and blood serum by ELISA techniques (Promega). Levels of NGF in the blood serum were significantly increased 8-fold at 48h but were significantly decreased in the spinal cord and DRG by 2- to 4-fold until 7 days postsurgery (ANOVA, p < 0.05). Contrary to accepted dogma, spinal injury results in decreased levels of NGF in the spinal cord and DRG following spinal injury.     

Endogenous neurotrophin-3 promotes neuronal sprouting from dorsal root ganglia

In the present study, we investigated the role of endogenous neurotrophin-3 in nerve terminal sprouting 2 months after spinal cord dorsal root rhizotomy. The left L_1–5 and L₇–S₂ dorsal root ganglia in adult cats were exposed and removed, preserving the L₆ dorsal root ganglia. Neurotrophin-3 was mainly expressed in large neurons in the dorsal root ganglia and in some neurons in spinal lamina II. Two months after rhizotomy, the number of neurotrophin-3-positive neurons in the spared dorsal root ganglia and the density of neurite sprouts emerging from these ganglia were increased. Intraperitoneal injection of an antibody against neurotrophin-3 decreased the density of neurite sprouts. These findings suggest that endogenous neurotrophin-3 is involved in spinal cord plasticity and regeneration, and that it promotes axonal sprouting from the dorsal root ganglia after spinal cord dorsal root rhizotomy.     

Visualizing sensory transmission between dorsal root ganglion and dorsal horn neurons in co-culture with calcium imaging

Sensory information is conveyed to the central nervous system by primary afferent neurons within dorsal root ganglia (DRG), which synapse onto neurons of the dorsal horn of the spinal cord. This synaptic connection is central to the processing of both sensory and pain stimuli. Here, we describe a model system to monitor synaptic transmission between DRG neurons and dorsal horn neurons that is compatible with high-throughput screening. This co-culture preparation comprises DRG and dorsal horn neurons and utilizes Ca(2+) imaging with the indicator dye Fura-2 to visualize synaptic transmission. Addition of capsaicin to co-cultures stimulated DRG neurons and led to activation of dorsal horn neurons as well as increased intracellular Ca(2+) concentrations. This effect was dose-dependent and absent when DRG neurons were omitted from the culture. NMDA receptors are a critical component of synapses between DRG and dorsal horn neurons as MK-801, a use-dependent non-competitive antagonist, prevented activation of dorsal horn neurons following capsaicin treatment. This model system allows for rapid and efficient analysis of noxious stimulus-evoked Ca(2+) signal transmission and provides a new approach both for investigating synaptic transmission in the spinal cord and for screening potential analgesic compounds.     

The role of neurotrophins in the maintenance of the spinal cord motor neurons and the dorsal root ganglia proprioceptive sensory neurons

The aim of this study was to approach the question of neuronal dependence on neurotrophins during embryonic development in mice in a way other than gene targeting. We employed amyogenic mouse embryos and fetuses that develop without any skeletal myoblasts or skeletal muscle and consequently lose motor and proprioceptive neurons. We hypothesized that if, in spite of the complete inability to maintain motor and proprioceptive neurons, the remaining spinal and dorsal root ganglia tissues of amyogenic fetuses still contain any of the neurotrophins, that particular neurotrophin alone is not sufficient for the maintenance of motor and proprioceptive neurons. Moreover, if the remaining spinal and dorsal root ganglia tissues still contain any of the neurotrophins, that particular neurotrophin alone may be sufficient for the maintenance of the remaining neurons (i.e., mostly non-muscle- and a few muscle-innervating neurons). To test the role of the spinal cord and dorsal root ganglia tissues in the maintenance of its neurons, we performed immunohistochemistry employing double-mutant and control tissues and antibodies against neurotrophins and their receptors. Our data suggested that: (a) during the peak of motor neuron cell death, the spinal cord and dorsal root ganglia distribution of neurotrophins was not altered; (b) the distribution of BDNF, NT-4/5, TrkB and TrkC, and not NT-3, was necessary for the maintenance of the spinal cord motor neurons; (c) the distribution of BDNF, NT-4/5 and TrkC, and not NT-3 and Trk B, was necessary for the maintenance of the DRG proprioceptive neurons; (d) NT-3 was responsible for the maintenance of the remaining neurons and glia in the spinal cord and dorsal root ganglia (possibly via TrkB).     

Neuronal overexpression of tissue-type plasminogen activator does not enhance sensory axon regeneration or locomotor recovery following dorsal hemisection of adult mouse thoracic spinal cord

CNS axons rarely regenerate spontaneously back to original targets following spinal cord injury (SCI). Neuronal expression of the serine protease tissue-type plasminogen activator (tPA) enhances axon growth in vitro and following PNS injury. Here we test the hypothesis that neuronal overexpression of tPA in adult transgenic mice promotes CNS axon regeneration and functional recovery following SCI. Adult wild-type and transgenic mouse spinal cords were subjected to dorsal hemisection at the level of the T10/T11 vertebrae. PCR confirmed incorporation of the transgene. Immunolabeling revealed overexpression of tPA in transgenic mice in neurons, including large-diameter neurons in lumbar dorsal root ganglia that contribute axons to the dorsal columns. Immunolabeling also revealed the presence of tPA protein within axons juxtaposing the injury site in transgenics but not wild types. In situ zymography revealed abundant enzymatic activity of tPA in gray matter of thoracic spinal cords of transgenics but not wild types. Rotorod locomotor testing revealed no differences between groups in locomotor function up to 21 days postinjury. Transganglionic tracer was injected into the crushed right sciatic nerve 28 days postinjury, and mice were killed 3 days later. There was no evidence for regrowth of ascending dorsal column sensory axons through or beyond the injury site. In conclusion, despite neuronal overexpression of tPA in injured neurons of transgenics, neither locomotor recovery nor regeneration of ascending sensory axons was observed following thoracic dorsal hemisection.     

Inflammation of rat dorsal root ganglia below a mid-thoracic spinal transection

Macrophages and T-lymphocytes invade the spinal cord in and around a lesion and spinal microglia are converted into macrophages. After spinal transection at T8 in rats, T-lymphocyte and major histocompatibility complex II+ (MHC II+) macrophage numbers were increased within dorsal root ganglia (DRGs) below the lesion. Inflammation was greater in DRGs closer to the site of transection. After 8 weeks, MHC II+cell density had fallen by 30% but T-lymphocyte numbers were undiminished. In lumbosacral DRGs, inflammation preceded inflammation within the spinal cord. The responses in distant DRGs are hard to reconcile with the limited damage to sensory neurons produced by the lesion. Inflammation of DRGs after spinal injury may contribute to hyper-reflexia and pain.     

GDNF and NGF family members and receptors in human fetal and adult spinal cord and dorsal root ganglia.

We describe the expression of mRNA encoding ligands and receptors of members of the GDNF family and members of the neurotrophin family in the adult human spinal cord and dorsal root ganglia (DRG). Fetal human spinal cord and ganglia were investigated for the presence of ligands and receptors of the neurotrophin family. Tissues were collected from human organ donors and after routine elective abortions. Messenger RNA was found encoding RET, GFR alpha-1, BDNF, trkB, and trkC in the adult human spinal cord and BDNF, NT-3, p75, trkB, and trkC in the fetal human spinal cord. The percentage of adult human DRG cells expressing p75, trkA, trkB, or trkC was 57, 46, 29, and 24%, respectively, and that of DRG cells expressing RET, GFR alpha-1, GFR alpha-2, or GFR alpha-3 was 79, 20, 51, and 32%, respectively. GFR alpha-2 was expressed selectively in small, GFR alpha-3 principally in small and GFR alpha-1 and RET in both large and small adult human DRG neurons. p75 and trkB were expressed by a wide range of DRG neurons while trkA was expressed in most small diameter and trkC primarily in large DRG neurons. Fetal DRG cells were positive for the same probes as adult DRG cells except for NT-3, which was only found in fetal DRG cells. Messenger RNA species only expressed at detectable levels in fetal but not adult spinal cord tissues included GDNF, GFR alpha-2, NT-3, and p75. Notably, GFR alpha-2, which is expressed in the adult rat spinal cord, was not found in the adult human spinal cord.     

Neuronal and glial expression of the adhesion molecule TAG-1 is regulated after peripheral nerve lesion or central neurodegeneration of adult nervous system

Expression of the cell adhesion molecule TAG-1 is down-regulated in adult brain, with the exception of certain areas exhibiting structural plasticity. Here, we present evidence that TAG-1 expression persists also in adult rat spinal cord and dorsal root ganglia (DRG), and can be up-regulated after injury. On Western blots of adult tissue, TAG-1 is detected as a 135-kDa band, with an additional specific 90-kDa band, not present in developing tissue. TAG-1 expression is found both in DRG neurons and in Schwann cells, particularly those associated with the peripherally projecting DRG processes. Quantitative in situ hybridization revealed that TAG-1 expression is significantly higher in small neurons that give rise to unmyelinated fibers, than in large DRG neurons. The regulation of TAG-1 was then examined in two different lesion paradigms. After a sciatic nerve lesion, TAG-1 expression is not up-regulated in DRG neurons, but decreases with time. At the lesion site, reactive Schwann cells up-regulate TAG-1, as demonstrated by both immunohistochemistry and in situ hybridization. In a second paradigm, we injected kainic acid into the spinal cord that kills neurons but spares glia and axons. TAG-1 is up-regulated in the spinal neuron-depleted area as well as in the corresponding dorsal and ventral roots, associated with both target-deprived afferent fibers and with the non-neuronal cells that invade the lesion site. These results demonstrate a local up-regulation of TAG-1 in the adult that is induced in response to injury, suggesting its involvement in axonal re-modelling, neuron-glia interactions, and glial cell migration.     

Cell death in the adult rat dorsal root ganglion after hind limb amputation, spinal cord transection, or both operations

Cell death of embryonic neurons which are unable to attain a proper target is well established. A delayed cell death of adult neurons permanently separated from their target tissue has been demonstrated for several cell groups. Cells of the dorsal root ganglia (DRG) are unique in that a single T-shape neurite has a peripheral branch which extends (for root L5) to the hind limb and a central branch extending into the spinal cord. We found a significant loss of L5 DRG neurons 25 weeks after hind limb amputation. This finding is consistent with the hypothesis that neuron survival is dependent on connection with a suitable target. We were unable to detect cell death in the DRG of L5 after complete spinal cord transection at T9. Separation of DRG cells from their central target is unimportant to neuronal survival.     

Effect of Brn-3a deficiency on CGRP-immunoreactivity in the dorsal root ganglion

Immunohistochemistry for calcitonin gene-related peptide (CGRP) was performed on the dorsal root ganglion (DRG) and spinal cord in wildtype and knockout mice for Brn-3a. CGRP-immunoreactive (-IR) neurons were abundant in the DRG of wildtype, heterozygous and knockout mice. Cell size analysis revealed that CGRP-IR neurons were of various sizes in wildtype and heterozygous mice. In the knockout mice, however, most of CGRP-IR neurons were small. In the spinal cord of knockout mice, the number of CGRP-IR fibers increased in the dorsal column but decreased in the deep part of the dorsal horn. The loss of Brn-3a may have different effects on CGRP-IR expression in small and large DRG neurons.     

GADD45A protects against cell death in dorsal root ganglion neurons following peripheral nerve injury

A significant loss of neurons in the dorsal root ganglia (DRG) has been reported in animal models of peripheral nerve injury. Neonatal sensory neurons are more susceptible than adult neurons to axotomy- or nerve growth factor (NGF) withdrawal-induced cell death. To develop therapies for preventing irreversible sensory cell loss, it is essential to understand the molecular mechanisms responsible for DRG cell death and survival. Here we describe how the expression of the growth arrest- and DNA damage-inducible gene 45α (GADD45A) is correlated with neuronal survival after axotomy in vivo and after NGF withdrawal in vitro. GADD45A expression is low at birth and does not change significantly after spinal nerve ligation (SNL). In contrast, GADD45A is robustly up-regulated in the adult rat DRG 24 hr after SNL, and this up-regulation persists as long as the injured fibers are prevented from regenerating. In vitro delivery of GADD45A protects neonatal rat DRG neurons from NGF withdrawal-induced cytochrome c release and cell death. In addition, in vivo knockdown of GADD45A expression in adult injured DRG by small hairpin RNA increased cell death. Our results indicate that GADD45A protects neuronal cells from SNL-induced cell death.     

Expression and localization of insulin receptor in rat dorsal root ganglion and spinal cord

The expression and localization of the insulin receptor (IR) was examined in rat dorsal root ganglia (DRG) and spinal cord using Western blotting, in situ hybridization and immunocytochemistry. Western blotting showed that the molecular weight of the IR beta subunit was higher in PNS than that found in CNS. Both IR mRNA and protein expressions were highest in small-sized sensory DRG neurons and myelinated sensory root fibers expressed higher levels of IR protein than myelinated anterior root fibers. In the spinal cord, IR immunoreactive neurons were present in lateral lamina V and in lamina X, suggesting the presence of IR in nociceptive pathways. Electronmicroscopy of DRGs revealed a polarized localization of the IR in abaxonal Schwann cell membranes, outer mesaxons in close vicinity to tight junctions of both myelinating and non-myelinating Schwann cells and to plasma membranes of sensory neurons. From these findings, we speculate that insulin may play a role in sensory fibers involved in nociceptive function often perturbed in diabetic neuropathy. The high expression of IR localizing to tight junctions of dorsal root mesaxons of DRGs may suggest a regulatory role on barrier functions compensating for the lack of a blood-nerve barrier in dorsal root ganglia. This is consistent with the colocalization of IR with tight junctions of the paranodal barrier and endoneurial endothelial cells in peripheral nerve.     

Centrifugal growth in orthotopic grafts of allogeneic dorsal root ganglia in adult rats: evidence for possible central ingrowth?

Fetal allogeneic dorsal root ganglion (DRG) transplants from 13-15 day rat embryo's (E13-E15) survived and differentiated when grafted orthopically (within the capsules of the excised 4th and 5th lumbar (L4-L5) ganglia) in adult rats. Survival of grafted neurones was established by prelabeling the grafts with a fluorescent vital dye (DiI) and visualizing the retained fluorescent marker 3 to 9 months later. Simultaneous retrograde tracing using fluorescent tracers applied in the spinal cord and peripheral nerve, respectively, yielded double-labeled dorsal root ganglion neurons, some of which were prelabeled. These findings demonstrate that prelabeled E13-E15 ganglia survive orthopic grafting, organotypically differentiate into mature DRG neurones, and can be double-labeled with fluorescent dyes applied to their peripherally and centrally directed processes. The presence of DiI containing cells which were retrogradely labeled from the spinal cord suggests that fetal (E13-E15) ganglia may have the capability of growing into a mature spinal cord.