Neutrophils invade lumbar dorsal root ganglia after chronic constriction injury of the sciatic nerve

To test whether neutrophils (PMN) target lumbar dorsal root ganglia (DRG) following axonal injury leading to neuropathic pain, we visualized PMN infiltration in DRG tissue sections and estimated PMN count by flow cytometry following sciatic chronic constriction injury (CCI). Seven days after CCI, results show PMN within DRG where their count increased by three fold ipsilateral to injury compared to contralateral or sham, concomitant with peak neuropathic pain behavior. Superoxide burst in PMN isolated from rats d7 after CCI was elevated by 170% +/-18 compared to na?ve and MCP-1 mRNA expression in DRG increased by 8.9+/-2.9 fold, but that of MIP-2, CINC-1, and RANTES did not change. We conclude that CCI causes PMN invasion of the DRG whereby the functional implication of their close proximity to neuronal axon and soma remains unknown.     

Thyroid hormone reduces the loss of axotomized sensory neurons in dorsal root ganglia after sciatic nerve transection in adult rat

We have shown that a local administration of thyroid hormones (T3) at the level of transected rat sciatic nerve induced a significant increase in the number of regenerated axons. To address the question of whether local administration of T3 rescues the axotomized sensory neurons from death, in the present study we estimated the total number of surviving neurons per dorsal root ganglion (DRG) in three experimental group animals. Forty-five days following rat sciatic nerve transection, the lumbar (L4 and L5) DRG were removed from PBS-control, T3-treated as well as from unoperated rats, and serial sections (1 microm) were cut. The physical dissector method was used to estimate the total number of sensory neurons in the DRGs. Our results revealed that in PBS-control rats transection of sciatic nerve leads to a significant (P < 0.001) decrease in the mean number of sensory neurons (8743.8 +/- 748.6) compared with the number of neurons in nontransected ganglion (mean 13,293.7 +/- 1368.4). However, administration of T3 immediately after sciatic nerve transection rescues a great number of axotomized neurons so that their mean neuron number (12,045.8 +/- 929.8) is not significantly different from the mean number of neurons in the nontransected ganglion. In addition, the volume of ganglia showed a similar tendency. These results suggest that T3 rescues a high number of axotomized sensory neurons from death and allows these cells to grow new axons. We believe that the relative preservation of neurons is important in considering future therapeutic approaches of human peripheral nerve lesion and sensory neuropathy.     

Changes in cholinergic and opioid receptors in the rat spinal cord,dorsal root and sciatic nerve after ventral and dorsal root lesion

Summary Changes in the distribution of³H-quinuclidinylbenzilate (³ H-QNB),³ H-acetylcholine (³ H-ACh) and³ H-alpha-bungarotoxin (alpha-BTx) binding sites were studied with the use of quantitative in vitro autoradiography in the L4–L6 segments of rats 7 days after ventral L4–L6-rhizotomies and 24 hours after ligation of the dorsal roots L4–L6. The changes in the binding sites of these ligands and of³ H-etorphine binding sites were also studied in the dorsal roots of the rats operated with dorsal root ligation and in the sciatic nerves (around a ligature) in the rats operated with ventral rhizotomy. After ventral rhizotomy³ H-QNB binding sites in the ipsilateral motor neuron area were decreased by about 25% from 100±5 to 73±5 fmol/mg wet weight. After dorsal root ligation³ H-QNB binding sites in the ipsilateral posterior horn were reduced by about 30% from 91±5 to 64±7 fmol/mg wet weight. No significant changes in the binding of the other cholinergic ligands in the spinal cords were observed after the operations. In the dorsal root³ H-alpha-Btx and³ H-etorphine binding sites were higher on the distal side of the ligation (3.5±0.8 and 14±4 fmol/mg wet weight, respectively) than on the proximal side (0.7±0.5 and 2.4±1.2 fmol/mg wet weight, respectively).The same level of³ H-ACh (total, muscarinic and nicotinic) binding was observed on both sides of the ligation. In the sciatic nerve³ H-QNB and total, muscarinic and nicotinic ACh binding sites were higher on the proximal side of the ligation than on the distal side. Except for a small emergence of muscarinic-ACh binding distally to the ligation there were no changes in the number of binding sites in the sciatic nerve after the ventral rhizotomy.Muscarinic antagonist binding sites are probably located on the perikarya of the motor neurons and presynaptically on the primary afferents in the posterior horn and in the dorsal root. Cholinergic agonist binding sites in the spinal cord seem less sensitive to axonal damage than antagonist binding sites. Cholinergic and opioid receptors in peripheral nerves are transported in both anterograde and retrograde directions and their origin seems to be the dorsal root ganglion.     

Regional distribution of substance P, neurokinin alpha and neurokinin beta in rat spinal cord, nerve roots and dorsal root ganglia, and the effects of dorsal root section or spinal transection

The regional distribution of 3 mammalian tachykinins (substance P, neurokinin alpha and neurokinin beta) in the rat spinal cord and related structures was investigated using a method of radioimmunoassay combined with high performance liquid chromatography. Substance P and neurokinin alpha were found to be distributed in a very similar manner with fairly constant molar ratios i.e. ratios of substance P to neurokinin alpha were 3.69 in the dorsal root ganglia, 3.49 in the dorsal root and 3.09 in the dorsal horn of the cervical spinal cord. On the other hand, the distribution of neurokinin beta was different from other tachykinins; although concentrated in the dorsal horn, neurokinin beta in the dorsal root ganglia or in the dorsal roots was negligibly small in amount. When the cervical dorsal roots were sectioned unilaterally, substance P and neurokinin alpha were decreased in a parallel fashion in the dorsal horn, whereas neurokinin beta was not. In addition neurokinin alpha was selectively and significantly decreased in the dorsal horn of the intact side when compared to that in the unoperated control rat. Since the magnitude of a decrease of neurokinin alpha in molar basis was approximately the same as a decrease of substance P, these findings suggest that the neurokinin alpha and substance P-containing primary afferent fibres could project partly to the contralateral dorsal horn as well. When the thoracic spinal cord was transected, substance P (and neurokinin alpha) was decreased in the ventral part of the lumbar spinal cord, suggesting the presence of tachykinin(s)-containing descending fibres.(ABSTRACT TRUNCATED AT 250 WORDS)     

Delayed loss of small dorsal root ganglion cells after transection of the rat sciatic nerve

The present study deals with changes in numbers and sizes of primary afferent neurons (dorsal root ganglion [DRG] cells) after sciatic nerve transection. We find that this lesion in adult rats leads to death of some DRG cells by 8 weeks and 37% by 32 weeks after the lesion. The loss of cells appears earlier in and is more severe in B-cells (small, dark cells with unmyelinated axons) than A-cells (large, light cells with myelinated axons). With regard to mean cell volumes, there is a tendency for both categories of DRG cells to be smaller, but except for isolated time points, these differences are not statistically significant. These findings differ from most earlier reports in that the cell loss takes place later than usually reported, that the loss is more severe for B-cells, and that neither A- or B-cells change size significantly. Accordingly, we conclude that sciatic nerve transection in adult rats leads to a slowly developing but relatively profound loss of primary afferent neurons that is more severe for B-cells. These results can serve as a basis for studies to determine the effectiveness of trophic or survival factors in avoiding axotomy induced cell death.     

trkB-like immunoreactivity in rat dorsal root ganglia following sciatic nerve injury

The expression of full-length trkB protein, the functional high affinity receptor for BDNF and NT-4, was examined by immunohistochemistry in adult rat L4–L5 dorsal root ganglia after different types of sciatic nerve lesions. In normal ganglia, 52.5% of the neurons showed trkB-like immunoreactivity. Size measurements demonstrated that trkB-like immunoreactivity was seen predominantly in small- and medium-sized cells. This was confirmed by the finding that 28% of all trkB-positive neurons showed affinity to RT97, an antibody which lanels a neurofilament epitope specific for medium-sized and large primary afferent neurons. After crush, section or neuroma formation of the sciatic nerve, the proportion of trkB-positive cells was 64.5%, 58% and 61.9%, respectively. Since trkB-receptors are present in regenerating primary afferent neurons, these data could indicate that BDNF and/or NT-4 are involved in sensory nerve fiber regeneration after adult injury.     

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.     

Changes in galanin immunoreactivity in rat lumbosacral spinal cord and dorsal root ganglia after spinal cord injury

Alterations in the expression of the neuropeptide galanin were examined in micturition reflex pathways 6 weeks after complete spinal cord transection (T8). In control animals, galanin expression was present in specific regions of the gray matter in the rostral lumbar and caudal lumbosacral spinal cord, including: (1) the dorsal commissure; (2) the superficial dorsal horn; (3) the regions of the intermediolateral cell column (L1-L2) and the sacral parasympathetic nucleus (L6-S1); and (4) the lateral collateral pathway in lumbosacral spinal segments. Densitometry analysis demonstrated significant increases (P < or = 0.001) in galanin immunoreactivity (IR) in these regions of the S1 spinal cord after spinal cord injury (SCI). Changes in galanin-IR were not observed at the L4-L6 segments except for an increase in galanin-IR in the dorsal commissure in the L4 segment. In contrast, decreases in galanin-IR were observed in the L1 segment. The number of galanin-IR cells increased (P < or = 0.001) in the L1 and S1 dorsal root ganglia (DRG) after SCI. In all DRG examined (L1, L2, L6, and S1), the percentage of bladder afferent cells expressing galanin-IR significantly increased (4-19-fold) after chronic SCI. In contrast, galanin expression in nerve fibers in the urinary bladder detrusor and urothelium was decreased or eliminated after SCI. Expression of the neurotrophic factors nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) was altered in the spinal cord after SCI. A significant increase in BDNF expression was present in spinal cord segments after SCI. In contrast, NGF expression was only increased in the spinal segments adjacent and rostral to the transection site (T7-T8), whereas spinal segments (T13-L1; L6-S1), distal to the transection site exhibited decreased NGF expression. Changes in galanin expression in micturition pathways after SCI may be mediated by changing neurotrophic factor expression, particularly BDNF. These changes may contribute to urinary bladder dysfunction after SCI.     

Temporal and spatial distribution of the aquaporin 1 in spinal cord and dorsal root ganglia after traumatic injuries of the sciatic nerve

Purpose

The aquaporin family comprises a large family of integral membrane proteins that enable the movement of water and other small, neutral solutes across plasma membranes. Although function and mechanism of aquaporins in central nervous system injury have been reported, the pathophysiologic role of aquaporin 1 (AQP1) in peripheral nerve has not been extensively documented. In the present study, we aimed to study the temporal and spatial distribution of AQP1 in spinal cord and dorsal root ganglia after sciatic nerve injury.

Methods

Forty-eight adult female mice were randomly divided into four groups (intact controls, sham operated, cut injury, and crush injury). Animals receiving cut or crush injuries were sacrificed at the 2nd, 24th, and 48th postoperative hours. Spinal cord samples at the level of lumbosacral intumescences and corresponding dorsal root ganglia on the experimental and contralateral side were dissected free and proceeded to AQP1 immunohistochemistry.

Results

Our quantitative estimations revealed that a sharp increase in AQP1 immunoreactivity at the 24th postoperative hour was observed. This sharp increase was no more evident at 48 h after sciatic nerve injury. Identical peak was observed after both cut and crush injuries.

Conclusions

We demonstrated that there was a temporal relationship with an increased expression of AQP1 following injury sustained to the sciatic nerve that was significantly observed in dorsal root ganglia and spinal cord. Those expressions were also subsided over time.     

Timing of c-jun protein induction in lumbar dorsal root ganglia after sciatic nerve transection varies with lesion distance

In situ hybridization and immunohistochemical studies have shown that sciatic nerve crush or transection induces upregulation of the immediate early gene c-jun mRNA and protein in lumbar dorsal root ganglion neurons. Here we have used enhanced chemiluminescent (ECL) immunoblotting as a sensitive and quantitative way of measuring the time course of c-jun protein induction following sciatic nerve transection at two distances from the L4 and L5 dorsal root ganglia. c-Jun protein was first detected within 3 h of proximal sciatic nerve transection and within 6 h of distal nerve transection. These results indicate substantially earlier increases in c-jun protein after nerve injury than previously reported, which can be attributed to the sensitivity of this detection method. The earlier induction of c-jun after proximal as compared to distal nerve transection supports the hypothesis that the c-jun response to sciatic nerve injury involves a distance-dependent signalling mechanism. © 1997 Elsevier Science B.V. All rights reserved.     

Identification and functional analysis of novel micro-RNAs in rat dorsal root ganglia after sciatic nerve resection

Peripheral nerve injures are quite common in clinical practice, and many studies have tried to explore the underlying molecular mechanisms. This study focuses on the identification and functional analysis of novel miRNAs in rat dorsal root ganglia (DRGs) following sciatic nerve resection, which is a classic model for studying peripheral nerve injury and regeneration. By using Solexa sequencing, computational analysis, Q-PCR verification, and Dicer knockdown assay, 114 novel miRNAs in rats were identified, of which 51 novel miRNAs were first reported in rat DRGs, and 63 novel miRNAs were produced at days 1, 4, 7, and 14 following sciatic nerve resection. We further predicted target genes of these miRNAs and analyzed the biological processes in which they were involved. The identified biological processes were consistent with the time frame of peripheral nerve injury and regeneration, revealing that these miRNAs were genuine miRNAs related to nerve regeneration. Our data provide an important resource for the future study of function and regulation of these miRNAs and contribute to elucidation of tyhe molecular mechanisms responsible for peripheral nerve injury and regeneration.     

Cell loss in lumbar dorsal root ganglia and transganglionic degeneration after sciatic nerve resection in the rat

The effects of sciatic nerve resection on lumbar dorsal root ganglion cells and their central branches have been studied in the adult rat. A quantitative analysis of the lumbar dorsal root ganglia indicated a 15–30% cell loss on the operated side. Argyrophilia indicating transganglionic degeneration was observed in Fink-Heimer stained sections from the lumbar spinal cord and the brainstem. The areas of degeneration argyrophilia were mainly located in the medial part of the ipsilateral L2–L6 dorsal horn laminae I–IV, the tract of Lissauer, the dorsal funiculus and the gracile nucleus. A few degenerating fibers could also be observed in the ipsilateral dorsal horn laminae V and VI, and in the ipsilateral ventral horn as well as in the contralateral dorsal and the gracile nucleus. The results confirm and extend previous findings at other levels and in other species. This suggests that cell loss and transganglionic degeneration may be general phenomena affecting a substantial proportion of primary sensory neurons following peripheral nerve injury.     

Distinct functional types of macrophage in dorsal root ganglia and spinal nerves proximal to sciatic and spinal nerve transections in the rat

Inflammation proximal to a peripheral nerve injury may be responsible for ectopic discharge and/or death of sensory neurones, factors thought to contribute to the development and/or maintenance of neuropathic pain. Here, ED1+, ED2+ and major histocompatibility complex class II (MHC II)+ macrophages in dorsal root ganglia (DRGs) and spinal nerve roots have been compared quantitatively in adult rats following transection of one sciatic or one spinal nerve, using double labelling immunohistochemistry. In control DRGs, all ED2+ cells expressed ED1 and some also MHC II. One week after either lesion, the ED2+ cells changed negligibly, except that all expressed MHC II. ED1+ and MHC II+ cell density increased markedly, with cells expressing MHC II alone (the majority), ED1/MHC II or rarely ED1 alone. In the spinal roots, ED1+ and MHC II+ cell density increased less after sciatic than after spinal nerve transection when ED1+ foamy cells were prominent. All ED2- macrophages were aggregated with T lymphocytes around blood vessels at 1 week or around isolated somata at later stages. ED1+ cell density declined more rapidly than MHC II+ cell density. Within the DRG, the debris of retrogradely labelled neurones appeared in ED2+ cells and a small proportion of MHC II+ cells that contained ED1. The data suggest that (i) resident ED2+ macrophages do not proliferate but are phagocytic and (ii) of ED1+ and MHC+ monocytes invading from the blood, only ED1+/MHC II+ cells are phagocytic. Four functional subtypes of macrophage within the DRGs were distinct from ED1+ foamy cells that phagocytosed myelin after spinal nerve transection.     

Comparison of c-jun,junB, and junD mRNA expression and protein in the rat dorsal root ganglia following sciatic nerve transection

The present study was designed to compare the expression of the Jun family of protooncogenes following nerve injury. Adult rats were anesthetized and the sciatic nerve transected. Dorsal root ganglia (DRG) at 1, 2, 3, and 7 days after nerve transection were collected, their total RNA extracted, and Northern blots performed using ³²P-labeled oligonucleotide probes. The constitutive expression of c-jun mRNA was very low in DRG. Induction of c jun mRNA was observed by day 1 after nerve transection, with a sixfold peak at 3 stays and a twofold induction still present by day 7. The constitutive expression of junB mRNA was also low in the DRG, and sciatic nerve transection produced only a modest induction (1.7fold by day 3) in the DRG ipsilateral to the nerve cut. junD mRNA was constitutively expressed at high levels in the DRG, and its level of expression did not change after sciatic nerve transection. Immunocytochemistry studies demonstrated a pattern of c-Jun, JunB, and JunD immunoreactivity (IR) associated with the cell nuclei of DRG neurons. c-Jun IR was found at very low levels in the undamaged contralateral DRG neurons, but sciatic nerve transection dramatically increased the number of c-Jun-immunoreactive neurons. Dot blot immunoblotting assay confirmed that the DRG ipsilateral to the sciatic nerve cut contained a higher level of c-Jun protein than the contralateral control DRG. Similar to c-Jun IR, JunB IR was minimal in the undamaged contralateral DRAG. However, the DRG ipsilateral to the nerve transection did not show an increase in the number of immunoreactive neurons. JunD protein was expressed at high levels in the contralateral DRG, and this level of expression persisted after sciatic nerve transection in the ipsilateral DRG. DNA gel retardation assay experiments with an AP-1 consensus sequence showed a single DNA-protein complex. This complex was increased in ipsilateral as compared with contralateral DRG extracts. The amount of DNA protein complex was reduced byc-Jun protein antiserum but was not altered when treated with a Fos antibody. We conclude that cjun, junB and junD mRNAs and proteins are differentially regulated in the DRG after sciatic nerve transection. © 1995 Wiley-Liss, Inc.     

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.     

Number of synapses increased in the rat spinal dorsal horn after sciatic nerve transection: a stereological study

We recently found that the number of synapses in the spinal dorsal horn, as estimated by stereological techniques, increased by 86% after chronic constriction injury of sciatic nerve in rats. In this study, we aimed to reveal whether transection of sciatic nerve was also associated with a plasticity change in the number of synapses. 18 adult SD rats were randomly divided into 3 groups undergoing (i) unilateral sham operation, (ii) unilateral sciatic nerve transection, and (iii) unilateral sciatic nerve transection with postoperative medication (parecoxib) for 3 days, respectively. 28 days postoperation, the L4-6 segment of the spinal cord was removed; paraffin-embedded sections were prepared and stained with Nissl's method and synaptophysin immunohistochemistry. The optical disector (a contemporary stereological technique) was used to estimate the numbers of neurons and synapses in the spinal dorsal horn. Compared to the non-operated side, the axotomy induced a 74.3% increase in the number of synapses per unit length of spinal cord or a 67.4% increase in the ratio between the numbers of synapses and neurons in the middle tissue block from the L4-6 segment on the operated side but not in either the rostral or caudal tissue block. Parecoxib had no effect on the parameters. In conclusion, peripheral nerve injury, model for neuropathic pain, is associated with a synaptic plasticity (numerical increase) in the spinal dorsal horn.