TGF-β Rescues Target-deprived Preganglionic Sympathetic Neurons in the Spinal Cord

Transforming growth factors β (TGF-β), a family of pleiotropic cytokines, are widely distributed in the developing and adult nervous system. In order to further determine the neural functions of TGF-β, we have localized the TGF-β isoforms 1, 2 and 3 in the adult rat adrenal medulla and studied the neuroprotective capacity of one representative family member, TGF-β2, for those spinal cord neurons which innervate adrenal chromaffin cells and which die after destruction of the adrenal medulla. Unilateral electrothermal destruction of the adrenal medulla led to the disappearance of 25% of sympathetic preganglionic neurons, which are located in the intermediolateral (IML) column of thoracic spinal cord segments 7–10 and can be selectively marked by NADPH-diaphorase. The neurons which disappeared following adrenomedullectomy constitute the full set of neurons that innervate the adrenal medulla. Implantation of gelfoam soaked with 0.5 μg TGF-β2 into the adrenal wound cavity rescued all spinal cord neurons in the IML ipsilaterally to the lesioned side. Cytochrome c was not effective. Injections of [¹²⁵l]TGF-β2 into the adrenal medulla did not result in retrograde transport and subsequent labelling of spinal cord neurons, suggesting that TGF-β may exert its neuroprotective actions by indirect mechanisms. TGF-β applied to cultured adrenocortical cells did not overtly increase the amount of mRNA for fibroblast growth factor-2, an established trophic molecule for sympathetic preganglionic spinal cord neurons. The mechanisms by which TGF-β exerts its neurotrophic effect are therefore unclear. Even so, our data provide the first evidence that TGF-β may play an important role in vivo in the control of maintenance of a population of spinal cord neurons.     

FGF-2 Is Sufficient to Isolate Progenitors Found in the Adult Mammalian Spinal Cord

The adult rat brain contains progenitor cells that can be induced to proliferatein vitroin response to FGF-2. In the present study we explored whether similar progenitor cells can be cultured from different levels (cervical, thoracic, lumbar, and sacral) of adult rat spinal cord and whether they give rise to neurons and glia as well as spinal cord-specific neurons (e.g., motoneurons). Cervical, thoracic, lumbar, and sacral areas of adult rat spinal cord (>3 months old) were microdissected and neural progenitors were isolated and cultured in serum-free medium containing FGF-2 (20 ng/ml) through multiple passages. Although all areas generated rapidly proliferating cells, the cultures were heterogeneous in nature and cell morphology varied within a given area as well as between areas. A percentage of cells from all areas of the spinal cord differentiate into cells displaying antigenic properties of neuronal, astroglial, and oligodendroglial lineages; however, the majority of cells from all regions expressed the immature proliferating progenitor marker vimentin. In established multipassage cultures, a few large, neuron-like cells expressed immunoreactivity for p75NGFr and did not express GFAP. These cells may be motoneurons. These results demonstrate that FGF-2 is mitogenic for progenitor cells from adult rat spinal cord that have the potential to give rise to glia and neurons including motoneurons.     

Leukaemia Inhibitory Factor or Related Factors Promote the Differentiation of Neuronal and Astrocytic Precursors within the Developing Murine Spinal Cord

Previously we have shown that leukaemia inhibitory factor (LIF) potentiates the development of murine spinal cord neurons in vitro , suggesting that it, or related factors, may play an important regulatory role in neuronal development. We have further investigated this role and show here that the generation of neurons in cultures of embryonic day 10 spinal cord cells is inhibited by antibodies to the β subunit of the LIF receptor. Since there are more undifferentiated precursors in antibody-treated cultures than in control and LIF-treated cultures, it is concluded that the primary action of LIF, or related molecules, is to promote neuronal differentiation, not precursor survival. In addition, the failure of LIF to support neuronal survival in the period immediately following differentiation suggests that the increased numbers of neurons generated with LIF are not attributable to its neurotrophic action. By selecting neuronal precursors on the basis of their inability to express class I major histocompatibility complex molecules, it was shown that LIF acted directly upon these cells and not via an intermediary cell. LIF also appears to be involved in regulating the differentiation of astrocytes, since it increases the number of glial fibrillary protein (GFAP)-positive cells present in the cultures and since the spontaneous production of GFAP-positive cells is blocked by antibodies to the LIF β receptor. These findings suggest that LIF or related factors promote the differentiation of neural precursors in the spinal cord, but that they are not involved in preferentially promoting precursors down a specific differentiation pathway.     

Membrane Characteristics and Synaptic Responsiveness of Superficial Dorsal Horn Neurons in a Slice Preparation of Adult Rat Spinal Cord

Intracellular recordings have been made from neurons of the superficial dorsal horn in slices of the lumbar and thoracic spinal cord of young adult rats. Three broad categories of neurons could be distinguished on the basis of their firing patterns to intracellular current pulses and their afterhyperpolarizations (AHP); there was no detectable difference in the regional distribution of the three types. Category 1 cells were characterized by maintained firing to intracellular depolarizing current pulses, brief action potential durations and polyphasic AHPs. Category 2 cells showed spike adaptation, without spike attenuation, during intracellular current pulses, and had monophasic AHPs. Category 3 cells fired only 1 or 2 spikes to maintained depolarizing pulses and had smaller monophasic AHPs than category 2 neurons. Spontaneous excitatory and inhibitory postsynaptic potential (epsp and ipsp) activity was seen with psp durations varying widely. Low intensity electrical stimulation of afferent fibres, or of superficial white matter, resulted in polyphasic epsps and/or ipsps. The spike discharge in response to such afferent inputs correlated with the membrane properties of the cells, such that the synaptic responses of category 1 neurons were usually bursts of spikes, whereas category 2 and 3 neurons either failed to fire or fired only a single spike. These results in adult rat spinal cord suggest that the discharge pattern within synaptic sensory responses of superficial dorsal horn neurons is determined by postsynaptic membrane properties as well as by the pattern of the afferent input.     

Expression of GAP-43 mRNA in Mouse Spinal Cord Following Unilateral Peripheral Nerve Damage: is There a Contralateral Effect?

Two new oligonucleotide anti-sense probes and their corresponding sense probes specific for mouse GAP-43 mRNA were synthesized and end-labelled with digoxygenin. They were used to localize GAP-43 mRNA in the spinal cords of normal mice and in mice 3 and 7 days following unilateral sciatic nerve cut. GAP-43 mRNA was found to be expressed at low levels in motor and other neurons of the normal spinal cords. As expected from other studies, up-regulation occurred in the cell bodies of axotomized motor neurons but, in addition, up-regulation was also observed in the cell bodies of intact motor neurons contralateral to the lesion. Densitometer measurements showed that the up-regulation of GAP-43 mRNA was less in the intact, contralateral motor neuron cell bodies than in the axotomized motor neuron cell bodies and furthermore was transient, being higher at 3 days than at 7 days following axotomy. Both anti-sense probes gave the same result, although differences in cellular localization were observed, and the two sense probes were negative. Probe binding was abolished by pretreatment of the sections with ribonuclease and hybridization was carried out under different conditions of stringency in order to ascertain whether the contralateral expression of GAP-43 mRNA was a true reflection of its distribution in vivo. There is conflicting evidence on the presence or absence of contralateral effects following unilateral peripheral nerve injury in the literature, and it is suggested that these differences can be accounted for by the methodology and type of probe used.     

Active and Passive Membrane Properties of Spinal Cord Neurons that Are Rhythmically Active during Swimming in Xenopus Embryos

Cellular properties have been examined in ventrally located Xenopus spinal cord neurons that are rhythmically active during fictive swimming and presumed to be motoneurons. Resting potentials and input resistances of such neurons are - 75 +/- 2 mV (mean +/- standard error) and 118 +/- 17 M ohm respectively. Most cells fire a single impulse, 0.5 to 2.0 ms in duration and 48.5 +/- 1.8 mV in amplitude, in response to a depolarizing current step. A minority fire several spikes of diminishing amplitude to more strongly depolarizing current. Cells held above spike, threshold fire on rebound from brief hyperpolarizing pulses. Spikes are blocked by 0.1 to 1.0 microM tetrodotoxin (TTX) and are therefore Na+-dependent. Current/voltage (I/V) plots to injected current are approximately linear near the resting potential but become non-linear at more depolarized levels. Cells recorded in TTX with CsCI-filled microelectrodes show a linearized I/V plot at depolarized membrane potentials suggesting the normal presence of a voltage-dependent K+ conductance activated at relatively depolarized levels. Most cells recorded in this way but without TTX fire long trains of spikes of near constant amplitude, pointing to a role of the K+ conductance in limiting firing in normal cells. Spike blockage with TTX reveals, in some cells, a transient depolarizing Cd2+-sensitive and therefore presumably Ca2+-dependent potential that increases in amplitude with depolarization. Cells in TTX, Cd2+, and strychnine, and recorded with CsCI-filled microelectrodes to block active conductances respond to hyperpolarizing current steps with a two component exponential response. The cell time constant (tau0) obtained from the longer of these by exponential peeling is relatively long (mean 15.7 ms). These findings contribute to an increased understanding of the cellular properties involved in spinal rhythm generation in this simple vertebrate.     

The Ability of Human Schwann Cell Grafts to Promote Regeneration in the Transected Nude Rat Spinal Cord

Advances in the purification and expansion of Schwann cells (SCs) from adult human peripheral nerve, together with biomaterials development, have made the construction of unique grafts with defined properties possible. We have utilized PAN/PVC guidance channels to form solid human SC grafts which can be transplanted either with or without the channel. We studied the ability of grafts placed with and without channels to support regeneration and to influence functional recovery; characteristics of the graft and host/graft interface were also compared. The T9–T10 spinal cord of nude rats was resected and a graft was placed across the gap; methylprednisolone was delivered acutely to decrease secondary injury. Channels minimized the immigration of connective tissue into grafts but contributed to some necrotic tissue loss, especially in the distal spinal cord. Grafts without channels contained more myelinated axons (x= 2129 ± 785) vs (x = 1442 ± 514) and were larger in cross-sectional area (x = 1.53 ± 0.24 mm²) vs (x= 0.95 ± 0.86 mm²). The interfaces formed between the host spinal cord and the grafts placed without channels were highly interdigitated and resembled CNS–PNS transition zones; chondroitin sulfate proteoglycans was deposited there. Whereas several neuronal populations including propriospinal, sensory, motoneuronal, and brainstem neurons regenerated into human SC grafts, only propriospinal and sensory neurons were observed to reenter the host spinal cord. Using combinations of anterograde and retrograde tracers, we observed regeneration of propriospinal neurons up to 2.6 mm beyond grafts. We estimate that 1% of the fibers that enter grafts reenter the host spinal cord by 45 days after grafting. Following retrograde tracing from the distal spinal cord, more labeled neurons were unexpectedly found in the region of the dextran amine anterograde tracer injection site where a marked inflammatory reaction had occurred. Animals with bridging grafts obtained modestly higher scores during open field [(x = 8.2 ± 0.35) vs (x = 6.8 ± 0.42),P = 0.02] and inclined plane testing (x = 38.6 ± 0.542) vs (x= 36.3 ± 0.53),P = 0.006] than animals with similar grafts in distally capped channels. In summary, this study showed that in the nude rat given methylprednisolone in combination with human SC grafts, some regenerative growth occurred beyond the graft and a modest improvement in function was observed.     

Neurons in the Paraventricular Nucleus of the Hypothalamus that Project to the Sexually Dimorphic Lower Lumbar Spinal Cord Concentrate 3H-Estradiol in the Male Rat

The location and distribution of estradiol-concentrating neurons in the hypothalamus afferent to segments of lumbar spinal cord that contain the sexually dimorphic spinal nucleus of the bulbocavernosus (SNB) were determined by combining retrograde fluorescent tract tracing with steroid hormone autoradiography. Injections of Fluorogold were made into segments of L5-L6 of the spinal cord of adult male rats and 12 days later animals were castrated. One week following castration, males received injections of [³H]estradiol and were perfused. Their brains were then processed for steroid hormone autoradiography. Following exposure times of 11 to 12 months, autoradiograms were developed and the hypothalamus was analyzed for neurons that concentrate estradiol and project to the spinal cord. Numerous neurons in the hypothalamus projected to the spinal cord, specifically neurons in the paraventricular nucleus (PVN), the lateral hypothalamus and the dorsal area of the hypothalamus. Although many subnuclei of PVN, as well as lateral hypothalamus, contained Fluorogold labelled neurons and estradiol concentrating neurons, the majority of double labeled cells were found in the lateral parvocellular (lp) subnucleus of PVN. Approximately 30% of the neurons in the lp subnucleus that projected to spinal cord also concentrated estradiol. Up to one half of the estradiol-concentrating neurons in Ip sent axons to the lower lumbar spinal cord. These results suggest that some of the effects of gonadal steroid hormones on SNB development, plasticity and function may in fact, be indirect, via steroid-sensitive afferents.     

Central Cord Syndrome of Cervical Spinal Cord Injury: Widespread Changes in Muscle Recruitment Studied by Voluntary Contractions and Transcranial Magnetic Stimulation

Muscle recruitment after central cord syndrome (CCS), a cervical spinal cord injury leading to a weaker motor function in the upper limbs versus the lower limbs, was examined in 14 individuals by means of voluntary muscle contractions and transcranial magnetic stimulation (TMS). Previously obtained data from able-bodied (AB) and non-CCS spinal cord injured subjects were used for comparison. Surface EMG was recorded from as many as six pairs of affected muscles. Individual muscle EMG activity was scored from 0 to 5. Cortical stimulation was applied while subjects maintained a weak contraction in each muscle. When CCS subjects attempted to produce a maximal voluntary contraction of an isolated muscle, this frequently resulted in cocontraction of nonsynergists in the same limb or/and in other limbs. Although the EMG scores in both upper and lower extremity muscles improved within postinjury time, in general, the lower extremity muscles, particularly the distal ones, demonstrated better recovery than the upper extremity muscles. CCS and AB subjects showed a similar high probability of “well-defined” responses to TMS (amplitude >150 μV) in all studied muscles. In contrast, latencies to TMS-evoked motor responses were prolonged by significant amounts after CCS. The delays in muscle responses were not significantly different from those observed in subjects with more severe cervical injury. Despite improvement in EMG scores, repeated measurements of TMS-evoked muscle response latencies in the same CCS subjects did not reveal significant shortening in central conduction latency. This argues against remyelination as an important contributor to the recovery process.     

不同年龄大鼠坐骨神经损伤后脊髓中神经生长导向因子Slit-1及Robo-2受体的表达变化

目的观察不同年龄大鼠坐骨神经损伤后,轴突导向因子Slit-1及其Robo-2受体在脊髓中的表达,以探讨不同年龄大鼠外周神经损伤后再生神经具有靶向性差异的可能机制。方法老年、成年和幼年大鼠各20只,建立左侧坐骨神经横断、硅胶管桥接模型。通过免疫荧光染色观察Slit-1蛋白和Robo-2受体在腰段脊髓中表达的变化,计算其荧光强度值,并进行统计学分析。结果伤后2周和4周,3组大鼠脊髓前角Slit蛋白均有较高表达,但各组间无显著差异。伤后2周和4周各组Robo-2受体表达均升高,其中老年鼠脊髓前角Robo-2受体表达明显高于成年和幼年组,差异有统计学意义(P0.05)。结论大鼠坐骨神经损伤后能刺激脊髓前角Slit-1高表达,不同年龄大鼠脊髓组织中Robo-2受体表达的差异可能决定了Slit-1在再生神经中的靶向性调节作用。     

Tonic,Burst, and Burst-Cycle Spinal Cord Stimulation Lead to Differential Brain Activation Patterns as Detected by Functional Magnetic Resonance Imaging

ObjectiveThe objective of this preclinical study was to examine the responses of the brain to noxious stimulation in the presence and absence of different modes of spinal cord stimulation (SCS) using blood-oxygen-level-dependent functional magnetic resonance imaging (BOLD-fMRI).Materials and MethodsSprague-Dawley rats were randomized to groups based on the mode of SCS delivered which included tonic stimulation (n = 27), burst stimulation (n = 30), and burst-cycle stimulation (n = 29). The control (sham) group (n = 28) received no SCS. The SCS electrode was inserted between T10 and T12 spinal levels prior to fMRI session. The experimental protocol for fMRI acquisition consisted of an initial noxious stimulation phase, a treatment phase wherein the SCS was turned on concurrently with noxious stimulation, and a residual effect phase wherein the noxious stimulation alone was turned on. The responses were statistically analyzed through paired t-test and the results were presented as z-scores for the quantitative analysis of the fMRI data.ResultsThe treatment with different SCS modes attenuated the BOLD brain responses to noxious hindlimb stimulation. The tonic, burst, and burst-cycle SCS treatment attenuated BOLD responses in the caudate putamen (CPu), insula (In), and secondary somatosensory cortex (S2). There was little to no corresponding change in sham control in these three regions. The burst and burst-cycle SCS demonstrated greater attenuation of BOLD signals in CPu, In, and S2 compared to tonic stimulation.ConclusionThe high-resolution fMRI study using a rat model demonstrated the potential of different SCS modes to act on several pain-matrix-related regions of the brain in response to noxious stimulation. The burst and burst-cycle SCS exhibited greater brain activity reduction in response to noxious hindlimb stimulation in the caudate putamen, insula, and secondary somatosensory cortex compared to tonic stimulation.     

Activation by Cutaneous or Visceral Noxious Stimulation of Spinal Neurons Projecting to the Medullary Dorsal Reticular Nucleus in the Rat: A c-fos Study

The involvement of spinal neurons in the transmission of cutaneous and visceral nociceptive input to the medullary dorsal reticular nucleus was studied. Rats were injected with cholera toxin subunit B in the left dorsal reticular nucleus and subjected 4 days later to noxious mechanical, thermal or chemical stimulation of the proximal internal aspect of the left thigh, or to chemical stimulation of the urinary bladder. Sections of spinal segments T₁₃-L₃ were processed immunocytochemically for cholera toxin subunit B and Fos protein. The percentage of double-labelled cells in the population of Fos-positive cells was higher in lamina I (1–4%) than in deeper laminae (0–0.7%) following all stimuli. The percentage of double-labelled cells in the population of retrogradely labelled cells was 30–53% in lamina I and 0–5% in laminae III—X. Visceral stimulation activated more retrogradely labelled lamina I cells than any kind of cutaneous stimulation. Pyramidal cells were activated in higher numbers than multipolar and flattened cells after thermal cutaneous or visceral stimulation, and in lower numbers than multipolar cells after mechanical stimulation. These results suggest that, in the experimental conditions used, spinal cord cells conveying noxious input to the dorsal reticular nucleus are concentrated in lamina I. They further indicate that the spinaldorsal reticular nucleus pathway plays a major role in the transmission of nociceptive visceral input, and point to the preferential involvement of pyramidal cells in cutaneous thermal and visceral processing.     

Tacrolimus (FK506) Increases Neuronal Expression of GAP-43 and Improves Functional Recovery after Spinal Cord Injury in Rats

Tacrolimus (FK506), a widely used immunosuppressant drug, has neurite-promoting activity in cultured PC12 cells and peripheral neurons. The present study investigated whether tacrolimus affects the expression of the neuronal growth-associated protein, GAP-43, as well as functional recovery after photothrombotic spinal cord injury in the rat. In injured animals receiving tacrolimus, the number of neurons expressing GAP-43 mRNA and protein approximately doubled compared to that in injured animals receiving vehicle alone. This increase in GAP-43-positive cells was paralleled by a significant improvement in neurological function evaluated by open-field and inclined plane tests. Another FKBP-12 ligand (V-10,367) had similar effects on GAP-43 expression and functional outcome, indicating that the observed effects of tacrolimus do not involve inhibition of the phosphatase calcineurin. Thus, tacrolimus, a drug which is already approved for use in humans, as well as other FKBP-12 ligands which do not inhibit calcineurin, could potentially enhance functional outcome after CNS injury in humans.     

Spinal cord injury induced heat shock protein expression is reduced by an antioxidant compound H-290/51. An experimental study using light and electron microscopy in the rat

Summary. The possibility that oxidative stress participates in heat shock protein 72 kD (HSP 72) expression following a focal trauma to the spinal cord was examined using a potent antioxidant compound H-290/51 in a rat model. A focal spinal cord injury (SCI) inflicted by making a longitudinal incision on the right dorsal horn of the T10–T11 segment under equithesin anaesthesia resulted in profound upregulation of HSP 72 expression in the adjacent spinal cord segments T9 and T12. This expression of HSP was most marked in the ipsilateral cord at 5 h after SCI. Pretreatment with H-290/51 (50 mg/kg, p.o.) 30 min before SCI markedly attenuated HSP expression in the spinal cord seen at 5 h. The motor functions of traumatized rats were also improved in the drug treated group. At this time, structural changes in the spinal cord and edema formation were considerable reduced compared to the untreated traumatized rats. Taken together, these observations suggest that (i) oxidative stress participates in HSP response following trauma, and (ii) the antioxidant compound H-290/51 attenuates cellularstress, improves motor functions and induces considerable neuroprotection in the early phase of SCI. Further studies using post-injury treatment with H-290/51 is needed to explore its therapeutic potentials in clinical settings.     

On the Distribution of GAP-43 and its Relation to Serotonin in Adult Monkey and Cat Spinal Cord and Lower Brainstem

By use of a monoclonal antibody, the distribution of growth-associated protein (GAP)-43-like immunoreactivity (LI) has been studied in the spinal cord of adult grey monkeys ( Macaca fascicularis ) and adult cats by use of immunofluorescence and peroxidase-antiperoxidase techniques. The brainstem was also studied with in situ hybridization histochemistry. In both monkeys and cats, a dense innervation of GAP-43-immunoreactive (IR) fibres was seen in close apposition to large cell bodies and their processes in the motor nucleus of the ventral horn. Double-labelling experiments revealed a high degree of coexistence between GAP-43- and 5-hydroxytryptamine (5-HT, serotonin)-LI in the monkey motor nucleus, while in the cat no such colocalization could be verified. At the electron microscopic level, GAP-43 labelling was seen as a coating of vesicles and axolemma inside the terminals. In both monkey and cat, cell bodies expressing mRNA encoding GAP-43 were demonstrated in the medullary midline raphe nuclei. A similar location was also encountered for mRNA for aromatic l -amino acid decarboxylase, an enzyme found in both catecholamine- and serotonin-containing neurons. The present results suggest that GAP-43 is present in the 5-HT bulbospinal pathway of the monkey. In the cat, GAP-43 mRNA-expressing cell bodies were demonstrated in areas where descending 5-HT neurons are located, but no convincing colocalization of 5-HT- and GAP-43-LI was found at spinal cord levels, despite the existence of extensive fibre networks containing either of the two compounds. Possible explanations for this species discrepancy are discussed. The function of GAP-43 in nerve terminals impinging on the motoneurons is unknown. However, it may play a role in transmitter release and/or plasticity, since such roles have been proposed for this protein in other systems.