Nerve growth factor in glia and inflammatory cells of the injured rat spinal cord

Nerve growth factor (NGF) is crucial for the development of sympathetic and small-diameter sensory neurons and for maintenance of their mature phenotype. Its role in generating neuronal pathophysiology is less well understood. After spinal cord injury, central processes of primary afferent fibers sprout into the dorsal horn, contributing to the development of autonomic dysfunctions and pain. NGF may promote these states as it stimulates sprouting of small-diameter afferent fibers and its concentration in the spinal cord increases after cord injury. The cells responsible for this increase must be identified to develop a strategy to prevent the afferent sprouting. Using immunocytochemistry, we identified cells containing NGF in spinal cord sections from intact rats and from rats 1 and 2 weeks after high thoracic cord transection. In intact rats, this neurotrophin was present in a few ramified microglia and in putative Schwann cells in the dorsal root. Within and close to the lesion of cord-injured rats, NGF was in many activated, ramified microglia, in a subset of astrocytes, and in small, round cells that were neither glia nor macrophages. NGF-immunoreactive putative Schwann cells were prevalent throughout the thoracolumbar cord in the dorsal roots and the dorsal root entry zones. Oligodendrocytes were never immunoreactive for this protein. Therapeutic strategies targeting spinal cord cells that produce NGF may prevent primary afferent sprouting and resulting clinical disorders after cord injury.     

病理性疼痛的分子机制

持续性或慢性疼痛是很多患者的主要描述症状。然而,现在的治疗手段还不能充分解决某些疼痛或会引起不能忍受的副作用。近来疼痛生物学者阐明了大量的参与疼痛发生和维持的细胞和分子活动。如何更好的理解这些分子活动的机制将有助于发展高效的,特异性的治疗手段。背根神经节中小细胞神经元向脊髓传递温觉和伤害性信息的感觉传递。这些神经元的外周突感受生理性和化学性刺激后,可以在脊髓背角的中枢突通过突触囊泡和大致密性囊泡释放兴奋性的神经递质和神经肽。这种兴奋性突触传递可以被一些抑制因子调控如脊髓中间神经元和下行系统中分泌的阿片肽、GABA、甘氨酸、5-羟色胺。本文将回顾脊髓抑制性系统所取得的一些研究进展,将重点介绍在阿片受体转运,阿片镇痛以及吗啡耐晋研究中的进展,这些发现可能的治疗前景也会一并讨论。     

Persistent At-Level Thermal Hyperalgesia and Tactile Allodynia Accompany Chronic Neuronal and Astrocyte Activation in Superficial Dorsal Horn following Mouse Cervical Contusion Spinal Cord Injury

In humans, sensory abnormalities, including neuropathic pain, often result from traumatic spinal cord injury (SCI). SCI can induce cellular changes in the CNS, termed central sensitization, that alter excitability of spinal cord neurons, including those in the dorsal horn involved in pain transmission. Persistently elevated levels of neuronal activity, glial activation, and glutamatergic transmission are thought to contribute to the hyperexcitability of these dorsal horn neurons, which can lead to maladaptive circuitry, aberrant pain processing and, ultimately, chronic neuropathic pain. Here we present a mouse model of SCI-induced neuropathic pain that exhibits a persistent pain phenotype accompanied by chronic neuronal hyperexcitability and glial activation in the spinal cord dorsal horn. We generated a unilateral cervical contusion injury at the C5 or C6 level of the adult mouse spinal cord. Following injury, an increase in the number of neurons expressing ΔFosB (a marker of chronic neuronal activation), persistent astrocyte activation and proliferation (as measured by GFAP and Ki67 expression), and a decrease in the expression of the astrocyte glutamate transporter GLT1 are observed in the ipsilateral superficial dorsal horn of cervical spinal cord. These changes have previously been associated with neuronal hyperexcitability and may contribute to altered pain transmission and chronic neuropathic pain. In our model, they are accompanied by robust at-level hyperaglesia in the ipsilateral forepaw and allodynia in both forepaws that are evident within two weeks following injury and persist for at least six weeks. Furthermore, the pain phenotype occurs in the absence of alterations in forelimb grip strength, suggesting that it represents sensory and not motor abnormalities. Given the importance of transgenic mouse technology, this clinically-relevant model provides a resource that can be used to study the molecular mechanisms contributing to neuropathic pain following SCI and to identify potential therapeutic targets for the treatment of chronic pathological pain.     

Recent evidence for activity-dependent initiation of sympathetic sprouting and neuropathic pain

Traumatic injury or inflammatory irritation of the peripheral nervous system often leads to persistent pathophysiological pain states. It has been well-documented that, after peripheral nerve injury or inflammation, functional and anatomical alterations sweep over the entire peripheral nervous system including the peripheral nerve endings, the injured or inflamed afferent fibers, the dorsal root ganglion (DRG), and the central afferent terminals in the spinal cord. Among all the changes, ectopic discharge or spontaneous activity of primary sensory neurons is of great clinical interest, as such discharges doubtless contribute to the develop-ment of pathological pain states such as neuropathic pain. Two key sources of abnormal spontaneous activity have been identified following peripheral nerve injury: the injured afferent fibers (neuroma) leading to the DRG, and the DRG somata. The purpose of this review is to provide a global account of the abnormal spontaneous activity in various animal models of pain. Particular attention is focused on the consequence of peripheral nerve injury and localized inflammation. Further, mechanisms involved in the generation of spontaneous activity are also reviewed; evidence of spontaneous activity in contributing to abnormal sympathetic sprouting in the axotomized DRG and to the initiation of neuropathic pain based on new findings from our research group are discussed. An improved understanding of the causes of spontaneous activity and the origins of neuropathic pain should facilitate the development of novel strategies for effective treatment of pathological pain.     

A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter connected to a radio transmitter into the femoral artery of rats that underwent a T4 spinal cord transection with or without grafting of embryonic brainstem-derived neural stem cells expressing green fluorescent protein. Compared to other methods such as cannula insertion or tail-cuff, telemetry is advantageous to continuously monitor blood pressure and heart rate in freely moving animals. It is also capable of long term multiple data acquisitions. In spinal cord injured rats, basal cardiovascular data under unrestrained condition and autonomic dysreflexia in response to colorectal distension were successfully recorded. In addition, cardiovascular parameters before and after SCI can be compared in the same rat if a transmitter is implanted before a spinal cord transection. One limitation of the described telemetry procedure is that implantation in the femoral artery may influence the blood supply to the ipsilateral hindlimb.     

Basal metabolic rate and autonomic nervous system dysfunction in men with spinal cord injury

Objectives: The purpose of this study was to determine the relationship between autonomic nervous system dysfunction and basal metabolic rate (BMR), and the effect of spasticity on basal metabolic rate. Research Method and Procedures: Twenty men (11 paraplegic and 9 tetraplegic) with American Spinal Injury Association (ASIA)‐A and ‐B grade chronic spinal cord injury (SCI) participated in this study. Total body fat mass and lean tissue mass were measured in all participants using DXA by standard methods. Patients were allocated into 2 groups to determine the effect of autonomic nervous system dysfunction on BMR: Group I (T6 and upper‐level injuries with history of autonomic dysreflexia) and Group II (T7 and lower‐level injuries without history of autonomic dysreflexia). Measurements of BMR were determined by indirect calorimetry under standardized conditions. Results: There were 13 patients in Group I and 7 patients in Group II and the difference between these two in terms of time since injury, BMI, age, weight, lean tissue mass, BMR, and BMR/kg were not significant. Conclusion: We concluded that autonomic nervous system dysfunction does not affect BMR, and it might be ignored in considering energy needs in spinal cord injury.     

Transplantation of Xenopus laevis ears reveals the ability to form afferent and efferent connections with the spinal cord

Previous comparative and developmental studies have suggested that the cholinergic inner ear efferent system derives from developmentally redirected facial branchial motor neurons that innervate the vertebrate ear hair cells instead of striated muscle fibers. Transplantation of Xenopus laevis ears into the path of spinal motor neuron axons could show whether spinal motor neurons could reroute to innervate the hair cells as efferent fibers. Such transplantations could also reveal whether ear development could occur in a novel location including afferent and efferent connections with the spinal cord. Ears from stage 24-26 embryos were transplanted from the head to the trunk and allowed to mature to stage 46. Of 109 transplanted ears, 73 developed with otoconia. The presence of hair cells was confirmed by specific markers and by general histology of the ear, including TEM. Injections of dyes ventral to the spinal cord revealed motor innervation of hair cells. This was confirmed by immunohistochemistry and by electron microscopy structural analysis, suggesting that some motor neurons rerouted to innervate the ear. Also, injection of dyes into the spinal cord labeled vestibular ganglion cells in transplanted ears indicating that these ganglion cells connected to the spinal cord. These nerves ran together with spinal nerves innervating the muscles, suggesting that fasciculation with existing fibers is necessary. Furthermore, ear removal had little effect on development of cranial and lateral line nerves. These results indicate that the ear can develop normally, in terms of histology, in a new location, complete with efferent and afferent innervations to and from the spinal cord.     

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of primary afferent fibers mediated by GABAergic axo-axonal synapses (primary afferent depolarization). The strength of primary afferent depolarization can be measured by recording of volume-conducted potentials at the dorsal root (dorsal root potentials, DRP). Pathological changes of presynaptic inhibition are crucial in the abnormal central processing of certain pain conditions and in some disorders of motor hyperexcitability. Here, we describe a method of recording DRP in vivo in mice. The preparation of spinal cord dorsal roots in the anesthetized animal and the recording procedure using suction electrodes are explained. This method allows measuring GABAergic DRP and thereby estimating spinal presynaptic inhibition in the living mouse. In combination with transgenic mouse models, DRP recording may serve as a powerful tool to investigate disease-associated spinal pathophysiology. In vivo recording has several advantages compared to ex vivo isolated spinal cord preparations, e.g. the possibility of simultaneous recording or manipulation of supraspinal networks and induction of DRP by stimulation of peripheral nerves.     

Pain and neurotransmitters

1. To study physiological roles of substance P (SP), gamma-aminobutyric acid (GABA), enkephalins and other endogenous substances, we developed several kinds of isolated spinal cord preparations of newborn rats. 2. In these preparations, various slow responses of spinal neurons evoked by stimulation of primary afferent C fibers were depressed by a tachykinin antagonist, spantide. These results together with many other lines of evidence suggest that SP and neurokinin A serve as pain transmitters in a subpopulation of primary afferent C fibers. 3. Some C-fiber responses in various isolated spinal cord preparations were depressed by GABA, muscimol, and opioid peptides. In contrast, bicuculline (GABA antagonist) and naloxone (opioid antagonist) potentiated the "tail pinch potential," i.e., a nociceptive response of the ventral root evoked by pinch stimulation of the tail in isolated spinal cord-tail preparation of the newborn rat. The latter results support the hypothesis that some primary afferents activate inhibitory spinal interneurons which release GABA and enkephalins as transmitters to modulate pain inputs.     

Speaking out of turn: a role for silent synapses in pain

Severe tissue or nerve injury can result in a chronic and inappropriate sensation of pain, mediated in part by the sensitization of spinal dorsal horn neurons to input from primary afferent fibers. Synaptic transmission at primary afferent synapses is mainly glutamatergic. Although a functioning excitatory synapse contains both alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors in the postsynaptic membrane, recent evidence suggests that dorsal horn neurons contain some "silent" synapses, which exhibit purely NMDA receptor-mediated evoked postsynaptic currents and do not conduct signals at resting membrane potential. Serotonin, which is released onto dorsal horn neurons by descending fibers from the rostroventral medulla, potentiates sensory transmission by activating silent synapses on those neurons, i.e., by recruiting functional AMPA receptors to the postsynaptic membrane. This phenomenon may contribute to the hyperexcitability of dorsal horn neurons seen in chronic pain conditions.     

Monoaminergic Modulation of Spinal Viscero-Sympathetic Function in the Neonatal Mouse Thoracic Spinal Cord

Descending serotonergic, noradrenergic, and dopaminergic systems project diffusely to sensory, motor and autonomic spinal cord regions. Using neonatal mice, this study examined monoaminergic modulation of visceral sensory input and sympathetic preganglionic output. Whole-cell recordings from sympathetic preganglionic neurons (SPNs) in spinal cord slice demonstrated that serotonin, noradrenaline, and dopamine modulated SPN excitability. Serotonin depolarized all, while noradrenaline and dopamine depolarized most SPNs. Serotonin and noradrenaline also increased SPN current-evoked firing frequency, while both increases and decreases were seen with dopamine. In an in vitro thoracolumbar spinal cord/sympathetic chain preparation, stimulation of splanchnic nerve visceral afferents evoked reflexes and subthreshold population synaptic potentials in thoracic ventral roots that were dose-dependently depressed by the monoamines. Visceral afferent stimulation also evoked bicuculline-sensitive dorsal root potentials thought to reflect presynaptic inhibition via primary afferent depolarization. These dorsal root potentials were likewise dose-dependently depressed by the monoamines. Concomitant monoaminergic depression of population afferent synaptic transmission recorded as dorsal horn field potentials was also seen. Collectively, serotonin, norepinephrine and dopamine were shown to exert broad and comparable modulatory regulation of viscero-sympathetic function. The general facilitation of SPN efferent excitability with simultaneous depression of visceral afferent-evoked motor output suggests that descending monoaminergic systems reconfigure spinal cord autonomic function away from visceral sensory influence. Coincident monoaminergic reductions in dorsal horn responses support a multifaceted modulatory shift in the encoding of spinal visceral afferent activity. Similar monoamine-induced changes have been observed for somatic sensorimotor function, suggesting an integrative modulatory response on spinal autonomic and somatic function.     

Endomorphin-2 is an endogenous opioid in primary sensory afferent fibers

Evidence is presented that the recently discovered endogenous mu-selective agonist, endomorphin-2, is localized in primary sensory afferents. Endomorphin-2-like immunoreactivity was found to be colocalized in a subset of substance P- and mu opiate receptor-containing fibers in the superficial laminae of the spinal cord and spinal trigeminal nucleus. Disruption of primary sensory afferents by mechanical (deafferentation by dorsal rhizotomy) or chemical (exposure to the primary afferent neurotoxin, capsaicin) methods virtually abolished endomorphin-2-like immunoreactivity in the dorsal horn. These results indicate that endomorphin-2 is present in primary afferent fibers where it can serve as the endogenous ligand for pre- and postsynaptic mu receptors and as a major modulator of pain perception.     

Cell transplantation therapies for spinal cord injury focusing on bone marrow mesenchymal stem cells: Advances and challenges

Spinal cord injury (SCI) is a devastating condition with complex pathological mechanisms that lead to sensory, motor, and autonomic dysfunction below the site of injury. To date, no effective therapy is available for the treatment of SCI. Recently, bone marrow-derived mesenchymal stem cells (BMMSCs) have been considered to be the most promising source for cellular therapies following SCI. The objective of the present review is to summarize the most recent insights into the cellular and molecular mechanism using BMMSC therapy to treat SCI. In this work, we review the specific mechanism of BMMSCs in SCI repair mainly from the following aspects: Neuroprotection, axon sprouting and/or regeneration, myelin regeneration, inhibitory microenvironments, glial scar formation, immunomodulation, and angiogenesis. Additionally, we summarize the latest evidence on the application of BMMSCs in clinical trials and further discuss the challenges and future directions for stem cell therapy in SCI models.     

Absence of Intraspinal Sprouting in Dorsal Root Axons Caudal to a Partial Spinal Hemisection: A Horseradish Peroxidase Transport Study

Primary afferent sprouting in the spinal cord was evaluated by comparing the central projection of horseradish peroxidase (HRP)-labeled sciatic nerve afferent axons in nonlesioned control rats, and in rats subjected to acute or chronic partial spinal hemisections as adults. The lesions were performed at various levels from T₁₀ to L₃, and removed supraspinal and varying amounts of descending propriospinal afferents to lumbar segments receiving the maximal sciatic projection. The hemisections typically involved all but the dorsal column, although in some cases a portion of the dorsal column, including the corticospinal tract, was also transected.

The distribution pattern and density of spinal HRP reaction product was not significantly different in experimental and control preparations in any segment below the lesion, regardless of the quantity of denervation, or the density of the normal sciatic projection in a given terminal region. These results, together with our previous finding concerning an absence of primary afferent sprouting following long-term dorsal root ganglionectomies, suggest that current concepts concerning collateral sprouting as a factor in functional plasticity in the mature mammalian spinal cord warrant re-evaluation.     

Spinal Astrogliosis in Pain Models: Cause and Effects

Pathological pain has been subjected to intense research to shed light on the underlying mechanisms of key symptoms, such as allodynia and hyperalgesia. The main focus has by and large concerned plasticity of spinal cord neurons and the primary afferent nerves relaying peripheral information to the spinal cord. Animal pain models display an increased presence of reactive astrocytes in the spinal cord, but in contrast to neurons, little is known about how they contribute to abnormal pain sensation. However, astrocytes are now beginning to receive greater attention, and as new information is emerging, it appears that astrocytes undertake critical roles in manifesting pathological pain. Through the secretion of diffusible transmitters, such as interleukins, ATP, and NO, astrocytes may augment primary afferent neuronal signaling or sensitize second order neurons in the spinal cord. In addition, astrocytes might lead to altered pain perception by a direct modulation of synaptic transmission between neurons in the nociceptive pathway or through the creation of astrocytic networks capable of transducing signals for extended distances across and along the spinal cord. Future research in astrocyte activation and signaling may therefore reveal novel drug targets for managing pathological pain.     

Analysis of the mechanisms of interaction between generators of cyclic motor reactions and inputs from suprasegmental systems

The simulation mathematical model of neuronal generator systems was used for analyzing the interaction between inputs from descending (afferent) systems and generators of scratching (locomotion). The data obtained indicate that in cases when generators of cyclic motor reactions influence the effectiveness of synaptic transmission from the fibers of descending (or afferent) systems, the differences in the main characteristics of signals that are produced by these generators themselves and those that come to them are emphasized. These data allow us to conclude that the system supplying interaction between inputs from suprasegmental (afferent) fiber systems and generators of locomotion of scratching can be interpreted as an adaptive filter which processes spatial and temporal information coming to the spinal cord via different suprasegmental or primary afferent inputs and allows generators of cyclic motor reactions to correct their functioning in accordance with changing external conditions.Neirofiziologiya/Neurophysiology, Vol. 25, No. 3, pp. 211–215, May–June, 1993.     

Migration patterns of sympathetic preganglionic neurons in embryonic rat spinal cord.

The displacement of immature neurons from their place of origin in the germinal epithelium toward their adult positions in the nervous system appears to involve migratory pathways or guides. While the importance of radial glial fibers in this process has long been recognized, data from recent investigations have suggested that other mechanisms might also play a role in directing the movement of young neurons. We have labeled autonomic preganglionic cells by microinjections of horseradish peroxidase (HRP) into the sympathetic chain ganglia of embryonic rats in order to study the migration and differentiation of these spinal cord neurons. Our results, in conjunction with previous observations, suggest that the migration pattern of preganglionic neurons can be divided into three distinct phases. In the first phase, the autonomic motor neurons arise in the ventral ventricular zone and migrate radially into the ventral horn of the developing spinal cord, where, together with somatic motor neurons, they form a single, primitive motor column (Phelps P. E., Barber R. P., and Vaughn J. E. (1991). J. Comp. Neurol. 307:77-86). During the second phase, the autonomic motor neurons separate from the somatic motor neurons and are displaced dorsally toward the intermediate spinal cord. When the preganglionic neurons reach the intermediolateral (IML) region, they become progressively more multipolar, and many of them undergo a change in alignment, from a dorsoventral to a mediolateral orientation. In the third phase of autonomic motor neuron development, some of these cells are displaced medially, and occupy sites between the IML and central canal. The primary and tertiary movements of the preganglionic neurons are in alignment with radial glial processes in the embryonic spinal cord, an arrangement that is consistent with a hypothesis that glial elements might guide autonomic motor neurons during these periods of development. In contrast, during the second phase, the dorsal translocation of preganglionic neurons occurs in an orientation perpendicular to radial glial fibers, indicating that glial elements are not involved in the secondary migration of these cells. The results of previous investigations have provided evidence that, in addition to glial processes, axonal pathways might provide a substrate for neuronal migration. Logically, therefore, it is possible that the secondary dorsolateral translocation of autonomic preganglionic neurons could be directed along early forming circumferential axons of spinal association interneurons, and this hypothesis is supported by the fact that such fibers are appropriately arrayed in both developmental time and space to guide this movement.     

Degeneration of primary afferent terminals following brachial plexus extensive avulsion injury in rats

Important breakthroughs in the understanding regeneration failure in an injured CNS have been made by studies of primary afferent neurons. Dorsal rhizotomy has provided an experimental model of brachial plexus (BP) avulsion. This is an injury in which the central branches of primary afferents are disrupted at their point of entry into the spinal cord, bringing motor and sensory dysfunction to the upper limbs. In the present work, the central axonal organization of primary afferents was examined in control (without lesion) adult Wistar rats and in rats subjected to a C3-T3 rhizotomy. Specific sensory axon subtypes were recognized by application of antibodies to the calcitonin gene-related peptide (CGRP), the P2X3 purinoreceptor, the low-affinity p75-neurotrophin receptor and the retrograde tracer cholera toxin subunit beta (TCbeta). Other subtypes weres labeled with the lectin Griffonia simplicifolia 1B4. Using immunohistochemistry and high resolution light microscopy, brachial plexus rhizotomy in adult rats has proven a reliable model for several neural deficits in humans. This lesion produced different degrees of terminal degeneration in the several types of primary afferents which define sub-populations of sensitive neurons. Between the C6 and C8 levels of the spinal cord,, deafferentation was partial for peptidergic GCRP-positive fibers, in contrast with elimination of non peptidergic and myelinated fibers. Dorsal rhizotomy has provided an adequate experimental model to study sensory alterations such as acute pain and allodynia as well as factors that affect regeneration into the CNS., Therefore, the differential deafferentation response must be considered inr the evaluation of therapies for nociception (pain) and regeneration for brachial plexus avulsion. The anatomical diffierences among the primary afferent subtypes also affect their roles in normal and damaged conditions.     

Refined sensory measures of neural repair in human spinal cord injury: bridging preclinical findings to clinical value

Sensory input from the periphery to the brain can be severely compromised or completely abolished after an injury to the spinal cord. Evidence from animal models suggests that endogenous repair processes in the spinal cord mediate extensive sprouting and that this might be further attenuated by targeted therapeutic interventions. However, the extent to which sprouting can contribute to spontaneous recovery after human spinal cord injury (SCI) remains largely unknown, in part because few measurement tools are available in order to non-invasively detect subtle changes in neurophysiology. The proposed application of segmental sensory evoked potentials (e.g., dermatomal contact heat evoked potentials and somatosensory evoked potentials) to assess conduction in ascending pathways (i.e., spinothalamic and dorsal column, respectively) differs from conventional approaches in that individual spinal segments adjacent to the level of lesion are examined. The adoption of these approaches into clinical research might provide improved resolution for measuring changes in sensory impairments and might determine the extent by which spontaneous recovery after SCI is mediated by similar endogenous repair mechanisms in humans as in animal models.     

Aromatase and 5-alpha reductase gene expression: modulation by pain and morphine treatment in male rats

Background

Neuronal transduction by adeno-associated viral (AAV) vectors has been demonstrated in cortex, brainstem, cerebellum, and sensory ganglia. Intrathecal delivery of AAV serotypes that transduce neurons in dorsal root ganglia (DRG) and spinal cord offers substantial opportunities to 1) further study mechanisms underlying chronic pain, and 2) develop novel gene-based therapies for the treatment and management of chronic pain using a non-invasive delivery route with established safety margins. In this study we have compared expression patterns of AAV serotype 5 (AAV5)- and AAV serotype 8 (AAV8)-mediated gene transfer to sensory neurons following intrathecal delivery by direct lumbar puncture.

Results

Intravenous mannitol pre-treatment significantly enhanced transduction of primary sensory neurons after direct lumbar puncture injection of AAV5 (rAAV5-GFP) or AAV8 (rAAV8-GFP) carrying the green fluorescent protein (GFP) gene. The presence of GFP in DRG neurons was consistent with the following evidence for primary afferent origin of the majority of GFP-positive fibers in spinal cord: 1) GFP-positive axons were evident in both dorsal roots and dorsal columns; and 2) dorsal rhizotomy, which severs the primary afferent input to spinal cord, abolished the majority of GFP labeling in dorsal horn. We found that both rAAV5-GFP and rAAV8-GFP appear to preferentially target large-diameter DRG neurons, while excluding the isolectin-B4 (IB4) -binding population of small diameter neurons. In addition, a larger proportion of CGRP-positive cells was transduced by rAAV5-GFP, compared to rAAV8-GFP.

Conclusions

The present study demonstrates the feasibility of minimally invasive gene transfer to sensory neurons using direct lumbar puncture and provides evidence for differential targeting of subtypes of DRG neurons by AAV vectors.