Analysis of Cutaneous Sensory Neurons in Transgenic Mice Lacking the Low Affinity Neurotrophin Receptor p75

Mice with a targeted mutation of the p75 low affinity neurotrophin receptor display smaller peripheral nerves and dorsal root ganglia. Here we show that transgenic mice have a significant elevation of thresholds to noxious mechanical and heat stimuli compared with p75+/+ control mice. Immunocytochemical analysis using antibodies against PGP 9.5 (a panaxonal marker) and calcitonin gene related peptide (CGRP, which labels peptidergic neurons) showed a reduction to 73% and 54%, respectively, of the epidermal innervation density. However, analysis of the cell size distribution of toluidine blue-stained dorsal root ganglia showed no selective loss of neurons of particular diameters. Moreover, the neurochemical profile of dorsal root ganglia cells as defined by trkA, CGRP, I84 and RT97 immunostaining revealed no significant differences in comparison with p75+/+ animals. Staining of the dorsal horn of the spinal cord for CGRP and 164 was also normal in p75-/-animals. Taking into account a previously reported loss of -50% dorsal root ganglion neurons, we conclude that all types of sensory neurons are equally depleted in p75-/- mice and that the absence of p75 impedes the development of more than one neuronal subtype.     

Regulation of Neurotrophin mRNA Expression in the Rat Brain by Glucocorticoids

Northern blot analysis was used to examine the effects of glucocorticoids on neurotrophin mRNA expression in the rat cerebral cortex and hippocampus. The results show that 3 days after adrenalectomy the mRNA levels for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) decreased significantly in both these regions. In adrenalectomized animals given dexamethasone replacement the mRNA levels for the three neurotrophins were restored to control levels. The effect of a single dose of dexamethasone (5 mg/kg) administered i p. to intact animals on the expression of neurotrophins was also examined. NGF and NT-3 mRNAs showed a 2.5-fold and a 1.4-fold increase, respectively, during the first 4 h after the injection. The increase was followed by a decrease, with levels -50% of control 24 and 48 h after the injection. In contrast, the level of BDNF mRNA did not change during the first 10 h after the injection, but decreased to 70% of control 48 h after the injection. These data indicate that glucocorticoids regulate neurotrophin mRNA expression both in the cortex and in the hippocampus, and suggest further that the known effects of glucocorticoids on neuronal survival in the brain could be due to changes in the levels of neurotrophins in the brain.     

Expression of c-Jun as a Response to Dorsal Root and Peripheral Nerve Section in Damaged and Adjacent Intact Primary Sensory Neurons in the Rat

It was previously shown that the immediate early gene, c-jun , was highly expressed over long periods, in both peripheral sensory and motor neurons following axon damage or block of axoplasmic transport. Here we have examined the question of whether the expression of c-Jun protein is related to axon injury per se or to the process of axon growth. We have examined dorsal root ganglion (DRG) cells subjected to different manipulations which are associated with varying degrees of regrowth, as follows: (i) after peripheral nerve section, where it appears that all damaged neurons make some regenerative effort. 1 – 24 days after sciatic nerve section and ligation most cells in L4/L5 DRG were c-Jun-positive; (ii) after section of the central processes of the DRG cells, which then showed a slow and limited regrowth of their axons towards, but not into, the spinal cord. This resulted in a variable, but significant, expression of c-Jun in a small number of DRG cells; (iii) in intact sensory neurons that were offered the opportunity to sprout into adjacent denervated peripheral tissue. The sciatic nerve was ligated and the response of cells in the L3 ganglia (many of which project to the saphenous nerve) was measured. A small but significant number of cells were c-Jun-positive; (iv) in intact sensory neurons that were offered the opportunity to sprout centrally into partialy denervated neuropil of the spinal cord. We examined neurons in the L3 DRG after rhizotomy of the adjacent L4/L5 dorsal roots. Previous work suggests that sensory neurons show at best a very limited growth under these conditions. No significant increase was seen in c-Jun expression in these cases. These results suggest that c-Jun expression is closely correlated with growth and regeneration, and not simply a consequence of neuronal injury.     

Positive Feedback between Acetylcholine and the Neurotrophins Nerve Growth Factor and Brain-derived Neurotrophic Factor in the Rat Hippocampus

In the rat hippocampus, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are synthesized by neurons in an activity-dependent manner. Glutamate receptor activation increases whereas GABAergic stimulation decreases NGF and BDNF mRNA levels. Here we demonstrate that NGF and BDNF mRNA and NGF protein are up-regulated in the rat hippocampus by the activation of muscarinic receptors. Conversely, NGF and BDNF enhance the release of acetylcholine (ACh) from rat hippocampal synaptosomes containing the nerve endings of the septal cholinergic neurons. NGF also rapidly increases the high-affinity choline transport into synaptosomes. The reciprocal regulation of ACh, NGF and BDNF in the hippocampus suggests a novel molecular framework by which the neurotrophins might influence synaptic plasticity.     

Different expressions of BDNF, NT3, and NT4 in muscle and nerve after various types of peripheral nerve injuries

The changes in the expression of brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4) in the rat neuromuscular system as a result of three different types of sciatic nerve injuries have been evaluated. The changes in mRNA and protein levels for BDNF, NT-3, and NT-4 in the soleus muscle and sciatic nerve were assessed 4-28 days after sciatic nerve transection (neurotmesis), sciatic nerve crush (axonotmesis), and mild acute compression (neurapraxia). BDNF mRNA levels increased dramatically with nerve transection in the soleus muscle and the sciatic nerve 7-14 days after injury, whereas the changes were low in other types of injury. The changes of protein levels for BDNF were also similar. The mRNA and the protein levels of NT-3 in the soleus muscle did not show any significant difference. The mRNA for NT-4 in the soleus muscle decreased from 4 to 14 days after sciatic nerve transection, and the protein level was also minimum 14 days after sciatic nerve transection. Our results indicate that the neurotrophic factors in the neuromuscular system could play a role in differentiating peripheral nerve injury.     

Developmentally Regulated Expression of HDNF/NT-3 mRNA in Rat Spinal Cord Motoneurons and Expression of BDNF mRNA in Embryonic Dorsal Root Ganglion

Northern blot analysis was used to demonstrate high levels of hippocampus-derived neurotrophic factor/neurotrophin-3 (HDNF/NT-3) mRNA in the embryonic day (E) 13 - 14 and 15 - 16 spinal cord. The level decreased at E18 - 19 and remained the same until postnatal day (P) 1, after which it decreased further to a level below the detection limit in the adult. In situ hybridization revealed that the NT-3 mRNA detected in the developing spinal cord was derived from motoneurons and the decrease seen at E18 - 19 was caused by a reduction in the number of motoneurons expressing NT-3 mRNA. The distribution of NT-3 mRNA-expressing cells in the E15 spinal cord was very similar to the distribution of cells expressing choline acetyltransferase or nerve growth factor receptor (NGFR) mRNA. Moreover, a striking similarity between the developmentally regulated expression of NT-3 and NGFR mRNA was noted in spinal cord motoneurons. A subpopulation of all neurons in the dorsal root ganglia expressed brain-derived neurotrophic factor (BDNF) mRNA from E13, the earliest time examined, to adulthood. These results are consistent with a trophic role of NT-3 for proprioceptive sensory neurons innervating the ventral horn, and imply a local action of BDNF for developing sensory neurons within the dorsal root ganglia.     

Three-Year Outcomes After Dorsal Root Ganglion Stimulation in the Treatment of Neuropathic Pain After Peripheral Nerve Injury of Upper and Lower Extremities

ObjectivesTraumatic peripheral nerve injuries (PNI) often result in severe neuropathic pain which typically becomes chronic, is recalcitrant to common analgesics, and is associated with sleep disturbances, anxiety, and depression. Pharmacological treatments proven to be effective against neuropathic pain are not well tolerated due to side effects. Neuromodulative interventions such as peripheral nerve or spinal cord stimulation have generated mixed results and may be limited by reduced somatotopic specificity. Dorsal root ganglion (DRG) stimulation may be more effective in this etiology.Materials and MethodsTwenty-seven patients were trialed with a DRG neurostimulation system for PNI; trial success (defined as ≥50% pain relief) was 85%, and 23 patients received a permanent stimulator. However, 36-month outcome data was only available for 21 patients. Pain, quality of life, mental and physical function, and opioid usage were assessed at baseline and at 3-, 6-, 12-, 18-, 24-, and 36 months post-permanent implant. Implant-related complications were also documented.ResultsCompared to baseline, we observed a significant pain relief (p < 0.001) at 3 (58%), 12 (66%), 18 (69%), 24 (71%), and 36 months (73%) in 21 patients (52.5 ± 14.2 years; 12 female), respectively. Mental and physical function showed immediate and sustained improvements. Participants reported improvements in quality of life. Opioid dosage reduced significantly (p < 0.001) at 3 (30%), 12 (93%), 18 (98%), 24 (99%), and 36 months (99%), and 20 of 21 patients were completely opioid-free after 36 months. There were five lead migrations and two electrode fractures (corrected by surgical intervention) and one wound infection (conservatively managed).ConclusionsDRG neuromodulation appears to be a safe, effective, and durable option for treating neuropathic pain caused by PNI. The treatment allows cessation of often ineffective pharmacotherapy (including opioid misuse) and significantly improves quality of life.     

Preferential Growth of Neonatal Rat Dorsal Root Ganglion Cells on Homotypic Peripheral Nerve Substrates In Vitro

Developing sensory neurons interact with molecular signals in the local environment to generate stereotypic nerve pathways. Regenerating neurons seem to lose the ability to reinnervate their original sites in the targets, resulting in abnormal sensory input and consequent clinical pathophysiology. The specificity of reinnervation of peripheral targets by regenerating axons is thus crucial for normal recovery of function. In this study, we have examined evidence for selectivity of interactions between primary afferent neurons from identified levels of the spinal cord and different peripheral nerve environments by culturing these neurons on sections of nerves to muscle and viscera. We have compared the growth of a population of sensory afferents normally innervating somatic targets (dorsal root ganglion cells from L4 and L5) with populations containing many afferents innervating visceral targets (L6 and S1 dorsal root ganglia and nodose ganglion). These neurons, from newly born rats, were cultured on unfixed cryostat sections of normal and prelesioned gastrocnemius nerve, pelvic spinal nerve and vagus nerve from adult rats. Normal muscle nerve was seen to support the regeneration of a significantly greater proportion of somatic neurons, with longer neurites, than the visceral nerves. Similarly, much higher proportions of the‘visceral’population of afferent neurons were seen to extend neurites on the normal visceral nerve substrates, with longer neurites, than on the muscle nerve substrate. The selectivity displayed by the sensory neurons for their normal nerve substrates was abolished when they were cultured on prelesioned nerve substrates subjected to Wallerian degeneration, which was apparent from the equivalent and increased proportions of growing neurons having comparable neurite lengths, on all the nerve substrates. We conclude that sensory neurons recognize and respond to substrate-specific and substrate-bound molecules present in normal adult peripheral nerves, and that these differences are lost in prelesioned nerves following Wallerian degeneration.     

Neurotrophin-3 prevents mitochondrial dysfunction in sensory neurons of streptozotocin-diabetic rats

Sensory neurons from streptozotocin (STZ)-diabetic rats exhibit depolarization of mitochondria and the related induction of reactive oxygen species has been proposed to contribute to the etiology of sensory polyneuropathy in diabetes. There is deficient neurotrophin-3 (NT-3)-dependent neurotrophic support of sensory neurons in diabetes and treatment of STZ-diabetic rats with NT-3 prevents neuropathological alterations in peripheral nerve. Therefore, we hypothesized that loss of NT-3 may contribute to mitochondrial dysfunction in sensory neurons in diabetic sensory neuropathy. The specific aim of this study was to determine whether treatment of STZ-diabetic rats with systemic NT-3 could prevent depolarization of the mitochondrial inner membrane potential (Deltapsi(m)). In vitro studies with cultured DRG neurons from control rats revealed that treatment with 50 ng/ml NT-3 for 6 h enhanced the Deltapsi(m), e.g., a higher polarized membrane potential, compared to untreated neurons (P < 0.05). Studies on DRG sensory neurons from control vs. STZ-diabetic rats demonstrated that NT-3 therapy prevented the diabetes-induced depolarization of Deltapsi(m) (P < 0.05) in parallel with normalization of diabetes-dependent deficits in sensory nerve conduction velocity. Furthermore, alterations in mitochondrial function in vitro and in vivo correlated with the level of activation/expression of Akt in DRG neurons.     

Analgesic Effects of Tonic and Burst Dorsal Root Ganglion Stimulation in Rats With Painful Tibial Nerve Injury

ObjectivesDorsal root ganglion (DRG) stimulation is effective in treating chronic pain. While burst stimulation has been proven to enhance the therapeutic efficacy in spinal cord stimulation, currently only a tonic stimulation waveform is clinically used in DRG stimulation. We hypothesized that burst DRG stimulation might also produce analgesic effect in a preclinical neuropathic pain model. We evaluated both the therapeutic effects of burst DRG stimulation and the possible effects of DRG stimulation upon inflammation within the DRG in a preclinical neuropathic pain model.Materials and MethodsRats received either a painful tibial nerve injury or sham surgery. Analgesic effects of DRG stimulation were evaluated by testing a battery of evoked pain-related behaviors as well as measuring the positive affective state associated with relief of spontaneous pain using conditioned place preference. Histological evidence for neuronal trauma or neuroinflammation was evaluated.ResultsAll of the waveforms tested (20 Hz-tonic, 20 Hz-burst, and 40 Hz-burst) have similar analgesic effects in sensory tests and conditioned place preference. Long-term DRG stimulation for two weeks does not change DRG expression of markers for nerve injury and neuroinflammation.ConclusionsDRG stimulation using burst waveform might be also suitable for treating neuropathic pain.     

Changes in Nuclear 3,5,3'-Triiodothyronine Receptor Expression in Rat Dorsal Root Ganglia and Sciatic Nerve During Development: Comparison with Regeneration

The action of the thyroid hormones on responsive cells in the peripheral nervous system requires the presence of nuclear triiodothyronine receptors (NT₃R). These nuclear receptors, including both the α and β subtypes of NT₃R, were visualized by immunocytochemistry with the specific 2B3 monoclonal antibody. In the dorsal root ganglia (DRG) of rat embryos, NT₃R immunoreactivity was first discretely revealed in a few neurons at embryonic day 14 (E14), then strongly expressed by all neurons at E17 and during the first postnatal week; all DRG neurons continued to possess clear NT₃R immunostaining, which faded slightly with age. The peripheral glial cells in the DRG displayed a short-lived NT₃R immunoreaction, starting at E17 and disappearing from the satellite and Schwann cells by postnatal days 3 and 7 respectively. In the developing sciatic nerve, Schwann cells also exhibited transient NT₃R immunoreactivity restricted to a short period ranging from E17 to postnatal day 10; the NT₃R immunostaining of the Schwann cells vanished proximodistally along the sciatic nerve, so that the Schwann cells rapidly became free of detectable NT₃R immunostaining. However, after the transection or crushing of an adult sciatic nerve, the NT₃R immunoreactivity reappeared in the Schwann cells adjacent to the lesion by 2 days, then along the distal segment in which the axons were degenerating, and finally disappeared by 45 days, when the regenerating axons were allowed to re-occupy the distal segment. It is concluded that (1) NT₃R expression lasts throughout the life of the DRG neurons; (2) NT₃R expression by peripheral glia is restricted to the perinatal period but may be transiently reactivated in Schwann cells after a nerve injury; and (3) cell-cell interaction with axons down-regulates the expression of NT₃R by Schwann cells in both growing and regenerating nerves.     

In Situ Hybridization of trkB and trkC Receptor mRNA in Rat Forebrain and Association with High-affinity Binding of [125I]BDNF, [125I]NT-4/5 and [125I]NT-3

The TrkB and TrkC receptor tyrosine kinases have been identified as high-affinity receptors for the neurotrophic factors brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5) and NT-3 respectively. These receptor classes were identified and mapped by the in situ hybridization of antisense riboprobes complementary to portions of the intracellular (tyrosine kinase) or extracellular (ligand-binding) domains of trkB and trkC mRNA, and by the distribution of high-affinity [¹²⁵I]BDNF, [¹²⁵I]NT-4/5 and [¹²⁵I]NT-3 binding sites in adjacent rat brain sections. Both methods showed that TrkB and TrkC receptors are abundant and widely expressed throughout the brain. Kinase or extracellular domain trkC probes labelled neuronal somata in a qualitatively similar manner in virtually every major area of the forebrain. Neither trkC probe labelled non-neuronal cells except for elements within cerebral arteries and arterioles. The kinase domain trkB probe hybridized exclusively to neurons. Neurons expressing trkB were even more widely distributed than those expressing trkC. The extracellular domain trkB probe labelled neurons with the same relative distribution as the trkB kinase domain probe, but also hybridized extensively with non-neural cells, particularly astrocytes, ependyma and choroid epithelium cells. The distribution of [¹²⁵I]NT-3 binding sites generally resembled that of trkC hybridization, particularly in the neocortex, striatum and thalamus. [¹²⁵I]BDNF and [¹²⁵I]NT-4/5 binding sites were more widely distributed and denser than those for [¹²⁵I]NT-3, and resembled the trkB hybridization pattern. These patterns are consistent with the preferential binding in the brain of TrkC receptors by [¹²⁵I]NT-3 and of TrkB receptors by [¹²⁵I]BDNF and [¹²⁵I]NT-4/5. That the predominantly neuronal patterns of hybridization obtained with kinase and extracellular domain probes for trkC are qualitatively indistinguishable suggests that truncated and full-length forms of TrkC are expressed within extensively overlapping populations of neurons. In marked contrast to TrkC, expression of the full-length and truncated forms of TrkB appears to be largely segregated, being expressed principally on neurons and non-neuronal cells respectively. The abundant and widespread neuronal distribution of full-length, signal-transducing forms of TrkB and TrkC predict that their cognate ligands, BDNF, NT-4/5 and NT-3, may exert direct effects on a large proportion of neurons within the mature brain.     

Dorsal Root Ganglion Stimulation for the Treatment of Non-Complex Regional Pain Syndrome Related Chronic Pain Syndromes: A Systematic Review

Background: While the majority of indications and approvals for dorsal root ganglion stimulation (DRGS) are for the refractory management of complex regional pain syndrome (CRPS), emerging evidence has suggested that DRGS may be favorably used for a plethora of other chronic pain phenomena. Consequently, we aimed to characterize the use and efficacy of DRGS for these non–CRPS-related chronic pain syndromes.Materials and Methods: A systematic review of clinical studies demonstrating the use of DRGS for non–CRPS-related chronic pain syndromes. The literature search was performed using PubMed, Cochrane Library, and CINAHL plus across August and September 2020.Results: A total of 28 reports comprising 354 total patients were included in the analysis. Of the chronic pain syndromes presented, axial low back pain, chronic pelvic and groin pain, other peripheral neuropathies, and studies with multiple concomitant pain syndromes, a majority demonstrated >50% mean pain reduction at the time of last follow-up following DRGS. Physical function, quality of life (QOL), and lesser pain medication usage also were repeatedly reported to be significantly improved.Conclusions: DRGS continues to lack supportive evidence from well designed, high level studies and recommendations from consensus committee experts. However, we present repeated and consistent evidence from lower level studies showing success with the use of DRGS for various non-CRPS chronic pain syndromes in reducing pain along with increasing function and QOL from one week to three years. Due to such low-level, high bias evidence, we strongly encourage the continuation of high-level studies in order to provide a stronger foundation for the use of DRGS in non-CRPS chronic pain patients. However, it may be reasonable and appropriate to evaluate patients for DRGS candidacy on a case-by-case basis particularly if they manifest focal pain syndromes refractory to noninterventional measures and may not be ideal candidates for other forms of neuromodulation.     

Neurotrophin and neuropeptide expression in mouse brain is regulated by knockout of the norepinephrine transporter

Neurotrophins [e.g. nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3)] and neuropeptides such as corticotropin-releasing factor (CRF) are reported to contribute to the action of antidepressants (ADs). Norepinephrine transporter (NET) knockout (NETKO) mice represent a model of chronic AD treatment. In the present study, we examined brain-region-specific regulations of NT-3, NGF, BDNF and CRF at the mRNA and protein level in NET wild-type (NETWT) and NETKO mice by means of quantitative real-time PCR (qPCR) and two-site enzyme-linked immunosorbent assays (ELISAs), respectively. NETKO-induced changes were detected for NT-3 in olfactory bulb, brainstem and whole brain at the mRNA and for olfactory bulb at the protein level, for NGF mRNA and protein in olfactory bulb, cerebellum and brainstem and for CRF mRNA and protein in the hippocampus. In contrast, BDNF levels remained unaltered. Our results suggest that NETKO mice represent a useful model to examine gene regulation of downstream targets potentially involved in the action of ADs. We could delineate NT-3, NGF and CRF as being regulated in distinct brain regions by KO of the NET.     

Prominent Perikaryal Expression of α- and β-Synuclein in Neurons of Dorsal Root Ganglion and in Medullary Neurons

Synucleins (syns) are a family of small, highly conserved proteins expressed predominantly in neurons. Although the normal function of syns is unknown, α-syn plays a pivotal role in several neurodegenerative diseases. The expression patterns of syns have been described in several studies, but much of this information was obtained before the cloning of all four members of this family of proteins and previous studies were limited to the analysis of single species. Here, we used antibodies specific for α-, β-, and γ-syn to study the patterns of expression in human, mouse, and rat nervous systems. Significant species-specific differences were detected in the expression of all three syns throughout the neuraxis. For example, γ-syn is highly expressed in human cortex, while it is present only at low levels in mouse and rat cortex. Moreover, in contrast to previous reports that α- and β-syns are normally localized predominantly at presynaptic terminals, we demonstrate that these proteins also are abundant in the perikarya of some neurons, such as in dorsal root ganglion. Intense α-syn immunoreactivity also was detected in the perikarya of human neurons in raphe, hypoglossal, and arcuate nuclei. These data underscore the need for additional studies to better understand the fundamental biological mechanism(s) targeting specific proteins to axonal terminals, as disruption of this process may be involved in the formation of pathological lesions.