Sprouting of primary afferent fibers after spinal cord transection in the rat

After spinal cord injury, hyper-reflexia can lead to episodic hypertension, muscle spasticity and urinary bladder dyssynergia. This condition may be caused by primary afferent fiber sprouting providing new input to partially denervated spinal interneurons, autonomic neurons and motor neurons. However, conflicting reports concerning afferent neurite sprouting after cord injury do not provide adequate information to associate sprouting with hyper-reflexia. Therefore, we studied the effect of mid-thoracic spinal cord transection on central projections of sensory neurons, quantified by area measurements. The area of myelinated afferent arbors, immunolabeled by cholera toxin B, was greater in laminae I-V in lumbar, but not thoracic cord, by one week after cord transection. Changes in small sensory neurons and their unmyelinated fibers, immunolabeled for calcitonin gene-related peptide, were assessed in the cord and in dorsal root ganglia. The area of calcitonin gene-related peptide-immunoreactive fibers in laminae III-V increased in all cord segments at two weeks after cord transection, but not at one week. Numbers of sensory neurons immunoreactive for calcitonin gene-related peptide were unchanged, suggesting that the increased area of immunoreactivity reflected sprouting rather than peptide up-regulation. Immunoreactive fibers in the lateral horn increased only above the lesion and in lumbar segments at two weeks after cord transection. They were not continuous with dorsal horn fibers, suggesting that they were not primary afferent fibers. Using the fluorescent tracer DiI to label afferent fibers, an increase in area could be seen in Clarke's nucleus caudal to the injury two weeks after transection. In conclusion, site- and time-dependent sprouting of myelinated and unmyelinated primary afferent fibers, and possibly interneurons, occurred after spinal cord transection. Afferent fiber sprouting did not reach autonomic or motor neurons directly, but may cause hyper-reflexia by increasing inputs to interneurons.     

Effect of spinal cord transection on N-methyl-D-aspartate receptors in the cord

Spinal cord injury can lead to an exaggeration of transmission through spinal pathways, resulting in muscle spasticity, chronic pain, and abnormal control of blood pressure and bladder function. These conditions are mediated, in part, by N-methyl-D-aspartate (NMDA) receptors on spinal neurons, but the effects of cord injury on the expression or function of these receptors is unknown. Therefore, antibodies to the NMDA-R1 receptor subunit and binding of [3H]MK-801 were used to assess NMDA receptors in the spinal cord. Receptor density in rats with intact spinal cords was compared to that in rats 1 and 2 weeks after spinal cord transection (SCT) at the mid-thoracic level. At 1 and 2 weeks after SCT, [3H]MK-801 binding was reduced in most laminae in cord segments caudal to the injury, whereas no decrease in amount of R1 subunit immunoreactivity was observed. No significant changes in [3H]MK-801 binding and NMDA-R1 immunoreactivity could be seen rostral to the transection. Since [3H]MK-801 binding requires an open ion channel, the discrepancy between [3H]MK-801 binding and immunocytochemistry may indicate a loss of functional receptors without a consistent change in their total number. Therefore, the exaggerated reflexes that are well established in rats 2 weeks after cord injury must be mediated by a mechanism that withstands attenuation of NMDA receptor function.     

Physiological functions of GABA-induced depolarizations in the developing rat spinal cord

Gamma-aminobutyric acid (GABA) is one of the principle inhibitory neurotransmitters in the mature spinal cord. It effectively suppresses synaptic transmission by mechanisms of postsynaptic and presynaptic inhibition. The function of GABA is less well understood early in spinal cord development, when the amino acid is transiently expressed in most neurons, and it depolarizes instead of hyperpolarizes neurons. This article reviews the possible physiological roles of GABA in modulating synaptic transmission, promoting neuronal development, and regulating neuronal pH during early stages of spinal cord differentiation. It is proposed that despite its depolarizing action, GABA acts as an inhibitory neurotransmitter that may also function as a neurotrophic agent.     

Effect of sciatic nerve transection or TTX application on enzyme activity in rat spinal cord

BACKGROUND/PURPOSE: Treatment of several congenital anomalies is frequently hindered by lack of enough tissue for surgical reconstruction in the neonatal period. The purposes of this study were (1) introduction of a novel concept in perinatal surgery, involving minimally invasive harvest of fetal tissue, which is then processed through tissue engineering techniques in vitro while pregnancy is allowed to continue, so that, at delivery, the newborn can benefit from having autologous, expanded tissue promptly available for surgical implantation at birth; (2) analysis of the progress of an engineered fetal skin graft with time, after implantation in the neonate; and (3) study of the effects of current tissue engineering techniques on fetal keratinocytes and fetal dermal fibroblasts. METHODS: Ten 90- to 95-day-gestation fetal lambs underwent surgical creation of two large paramedian excisional skin defects on the posterior body wall. Subsequently, fetal skin specimens no larger than 1.5 x 1.5 cm were videofetoscopically harvested. Fetal keratinocytes and dermal fibroblasts were then separately cultivated and expanded in vitro for 45 to 50 days, resulting in a total of approximately 250 to 300 million cells. Seven to 10 days before fetal delivery, all cells were seeded in two layers on a 16 to 20-cm2, 3-mm thick biodegradable polyglycolic acid polymer matrix. One to 4 days after delivery, the autologous engineered skin was implanted over one of two previously created skin defects. The second skin defect region received an absorbable polymer scaffold without cells as a control. If necessary, the original skin wounds were further amplified before implantation. Each animal provided at least one time-point for histological analysis of both types of repair through excisional biopsies performed at weekly intervals, up to 8 weeks postimplantation. Normal skin specimens were also used as controls. RESULTS: Fetal and neonatal survival rates were 100%. Based on previous postnatal skin engineering studies, fetal dermal fibroblasts multiplied significantly faster in vitro (approximately fivefold) than expected. Fetal keratinocytes multiplied at expected postnatal rates. The engineered grafts induced faster epithelization of the wound (partial at 1 week and complete between 2 and 3 weeks postoperatively) than did the acellular ones (partial at 3 weeks and complete between 3 and 4 weeks postoperatively). Analysis of skin architecture showed a higher level of epidermal organization and less dermal scarring in the wounds that received the engineered, cell-implanted polymer scaffold. CONCLUSIONS: (1) Videofetoscopically assisted fetal tissue engineering is a viable method for obtaining expanded autologous tissue for prompt surgical reconstruction at birth. (2) Fetal skin can be expanded and engineered in vitro at faster rates than expected postnatally, with current tissue engineering techniques. (3) Engineered autologous fetal skin induces a faster and more organized healing of neonatal skin defects than that observed with second intention. This concept may prove useful for the treatment of certain human neonatal conditions such as giant neoplasias, ectopia cordis, and other body wall defects.     

Retinoid-X receptor signalling in the developing spinal cord

Retinoids regulate gene expression through the action of retinoic acid receptors (RARs) and retinoid-X receptors (RXRs), which both belong to the family of nuclear hormone receptors. Retinoids are of fundamental importance during development, but it has been difficult to assess the distribution of ligand-activated receptors in vivo. This is particularly the case for RXR, which is a critical unliganded auxiliary protein for several nuclear receptors, including RAR, but its ligand-activated role in vivo remains uncertain. Here we describe an assay in transgenic mice, based on the expression of an effector fusion protein linking the ligand-binding domain of either RXR or RAR to the yeast Gal4 DNA-binding domain, and the in situ detection of ligand-activated effector proteins by using an inducible transgenic lacZ reporter gene. We detect receptor activation in the spinal cord in a pattern that indicates that the receptor functions in the maturation of limb-innervating motor neurons. Our results reveal a specific activation pattern of Gal4-RXR which indicates that RXR is a critical bona fide receptor in the developing spinal cord.     

Angiotensin II in the spinal cord of the rat and its sympatho-excitatory effects

Immunohistochemical studies have shown there is a dense angiotensin-like immunoreactivity of terminals in the sympathetic region of the thoracic and lumbar spinal cord. In the present study measurements were made of the concentration of angiotensin in the spinal cord of rats using radioimmunoassay following two different extraction procedures. These gave concentrations of angiotensin as mean of 108 and 161 pg.g-1 tissue wet weight. Angiotensin II given intrathecally or microinjected into the spinal cord caused an increase in postganglionic sympathetic nerve activity which was blocked by prior application of saralasin. Angiotensin III was without effect. Intracellular recordings from sympathetic preganglionic neurones in-vitro in slices of neonate rat spinal cord showed that angiotensin II produced an increase of excitability of the neurones by a slow depolarisation without the generation of action potentials. This effect still occurred in the presence of TTX. Angiotensin II also could increase synaptic activity, both EPSPs and IPSPs as well as a synaptically induced slow depolarisation being observed suggesting that presympathetic interneurones are also sensitive to the peptide. The evidence indicates that if angiotensin is released from nerve terminals surrounding sympathetic neurones it will enhance the gain of the neurone so that it could more easily be discharged by other excitatory inputs.     

The effects of interleukin-1 receptor antagonist protein (IRAP) infusion following spinal cord transection in rats

A laminectomy was performed at the T5-T6 vertebral level in adult, male, Sprague-Dawley rats and the spinal cord transected with a scalpel. A group of sham animals was subjected to the same surgery without the transection step. A group of unhandled control rats was also included. A subgroup of transected animals received a subcutaneous osmotic minipump that dispensed IL-1 receptor antagonist protein (IRAP) at the transection site for 7 consecutive days. Another transected subgroup received a minipump that infused the vehicle only. IRAP-treated rats displayed a significant reduction in body temperature (p < 0.05) compared with vehicle-treated rats. The IRAP-treated rats were also less active when assessed for locomotor behavior using an HVS computerized tracking system (p < 0.01). IRAP treatment had no effect on serum corticosterone, beta-endorphin levels, Con A, PHA, or LPS-induced splenocyte mitogenesis when compared with vehicle-treated animals. However, half of the IRAP-treated animals exhibited a substantive reduction in the number of reactive astrocytes near the transection site, suggesting a possible effect of IRAP on astrocyte activation.     

Peripheral and central contributions to the persistent expression of spinal cord fos-like immunoreactivity produced by sciatic nerve transection in the rat

In the absence of reliable baseline data for normal neuron density in the intestine, the diagnosis of hypo- and hyperganglionosis is purely subjective. This study has established the normal neuron density by neuron counts in paraffin sections taken both transversely (transverse sections, TS) and longitudinally (longitudinally sections, LS) in relation to the long axis of normal postmortem jejunum, ileum, and colon from 21 children (aged 4 weeks to 10 years). Intestine from two adults (aged 16 and 42 years) and colon alone from a further six adults (aged 16 to 83 years) were also studied. The mean density values in childhood were for jejunum 3.6/mm (TS), 3.7/mm (LS); for ileum 4.3/mm (TS, LS); and for colon 7/mm (LS), 7.7/mm (TS). The proximal margins of surgically resected colons from six patients with Hirschsprung's disease and one patient with suspected isolated hypoganglionosis were also analyzed and the neuron densities compared with the established postmortem data. Neuron density values outside two standard deviations from the postmortem mean were shown to correlate with continuing pseudo-obstructive symptoms in these patients.     

Ultrastructural changes in the rat phrenic nucleus developing within 2 h after cervical spinal cord hemisection

The present study was conducted to describe the ultrastructural changes which occur in the young adult rat phrenic nucleus within 2 h after an ipsilateral C2 spinal cord hemisection. The main objective was to determine if there is a temporal relationship between specific ultrastructural changes in the phrenic nucleus and a significant augmentation of crossed phrenic nerve activity which occurs as early as 2 h after hemisection. Phrenic motoneurons were identified at electron microscopic levels by retrograde HRP labeling. Ultrastructural features in the phrenic nucleus of control and experimental rats were qualitatively analyzed and then quantitated. At 2 h posthemisection, there was a significant increase in the mean percentage of phrenic dendrodendritic appositions. In the control rats, 4.73 +/- 0.18% of phrenic dendrites were in apposition, and this percentage increased significantly to 8.58 +/- 0.54% at 2 h after injury. Furthermore, the mean lengths of asymmetrical and symmetrical synaptic active zones increased significantly at 2 h posthemisection from control lengths of 0.372 +/- 0.009 microns and 0.404 +/- 0.007 microns to 0.410 +/- 0.011 microns and 0.513 +/- 0.032 microns, respectively, in experimental rats. The phrenic nucleus is therefore capable of morphological plasticity as early as 2 h after spinal cord hemisection and this plasticity coincides temporally with the physiological augmentation of crossed phrenic nerve activity at 2 h. The data further suggest that these morphological changes may be part of the substrate for the unmasking of ineffective synapses during the crossed phrenic phenomenon.     

Instrumental learning within the spinal cord: I. Behavioral properties

Four experiments are reported that explore whether spinal neurons can support instrumental learning. During training, one group of spinal rats (master) received legshock whenever one hindlimb was extended. Another group (yoked) received legshock independent of leg position. Master, but not yoked, rats learned to maintain their leg in a flexed position, exhibiting progressively longer flexions as a function of training (Experiment 1). All subjects were then tested by applying controllable shock to the same leg (Experiment 2). Master rats reacquired the instrumental response more rapidly (positive transfer), whereas yoked rats failed to learn (a learned helplessness-like effect). Disrupting response-outcome contiguity by delaying the onset and offset of shock by 100 ms eliminated learning (Experiment 3). Experiment 4 showed that shock onset contributes more to learning than does shock offset.     

Postnatal growth of corticospinal axons in the spinal cord of developing mice

The corticospinal tract (CST) plays an important role in the control of voluntary movements. Although the development of the CST has been studied extensively in other species, limited information is available on its development in mice. In the present study, the growth of corticospinal axons was characterized in developing mice using Phaseolus vulgaris leucoagglutinin (PHA-L). Our results indicate that the leading CST axons reach the 8th cervical segment at postnatal day (PD) 2, the 7th thoracic segment at PD4, the 13th thoracic segment at PD7, and the 5th lumbar segment at PD9. The arrival of corticospinal axons at the distal lumbar cord at PD9 was further confirmed by retrograde tracing using fast blue (FB). A waiting period of 2-3 days exists after the leading CST axons pass a particular segment before sending collaterals into the gray matter of that segment. The CST continues to increase in size in lower thoracic and lumbar areas up to PD14 when its adult appearance is achieved. In this study, the date of animal's sacrifice was used as the specific postnatal date to demonstrate the growth of the CST. This definition gives a more reliable indication of the exact location of the CST at a specific developmental time point since the CST continues to grow after tracer injections and since the dye is transported much faster than axonal growth. We suggest that these findings can be used as a template for studies on both normal and transgenic mice where some developmental significance is given to the CST.     

Morphology and position of growth cones in the developing Xenopus spinal cord

Axonal growth cones in longitudinal fiber tracts of the developing spinal cord of Xenopus were examined using electron microscopy. Fiber tracts of the spinal cord develop by the ingrowth of fibers, into pre-existing longitudinally oriented spaces between adjacent neuroepithelial cells of the neural tube. Growth cones seen among the neurites of the tracts were identified by their generally larger size (1.2 X 4.5 micrometer), bulbous and irregular outlines, and cytoplasmic components. Overall cytoplasmic density was usually less than that of surrounding neuroepithelial cells and axons. They contained few organelles, among them assorted clear and densecored vesicles, agranular reticulum, and occasional mitochondria and autographic vacuoles. Microtubules were rarely present. Growth cones appeared to conform in outline to the space which they occupied. Smaller extensions which resembled the filopodia described by others insinuated themselves among other elements of the fiber fascicles. The filopodia contained a fine granular or filamentous feltwork. Growth cones consistently appeared at the interface of other axons in the fascicle and the peripheral neuroepithelial endfeet. In longitudinal sections of fascicles containing more than one growth cone, the growth cones were layered in a pattern suggesting that new cones are added by pushing between the next youngest fibers and the peripheral neuroepithelial processes of the cord. The possible significance of this finding in the achievement of order in the spinal tracts is discussed.     

Behavioral effects of milk in the rat fetus.

Intraoral infusion of milk to the rat fetus promoted changes in behavior (mouth and rearlimb movements), reduced responsiveness to perioral cutaneous stimulation, and resulted in expression of a fetal stretch response. Milk also altered the temporal organization of fetal movements over periods up to 30 min. The orosensory characteristics of milk, in the absence of ingestion, was sufficient to evoke these behavioral effects. Reduced responsiveness to a perioral stimulus had a rapid onset (     

Development of spontaneous synaptic transmission in the rat spinal cord

Dorsal root afferents form synaptic connections on motoneurons a few days after motoneuron clustering in the rat lumbar spinal cord, but frequent spontaneous synaptic potentials are detected only after birth. To increase our understanding of the mechanisms underlying the differentiation of synaptic transmission, we examined the developmental changes in properties of spontaneous synaptic transmission at early stages of synapse formation. Spontaneous postsynaptic currents (PSCs) and tetrodotoxin (TTX)-resistant miniature PSCs (mPSCs) were measured in spinal motoneurons of embryonic and postnatal rats using whole cell patch-clamp recordings. Spontaneous PSC frequencies were higher than mPSC frequencies in both embryonic and postnatal motoneurons, suggesting that even at embryonic stages, when action-potential firing rate was low, presynaptic action potentials played an important role in triggering spontaneous PSCs. After birth, the twofold increase in spontaneous PSC frequency was attributed to an increase in action-potential-independent quantal release rather than to a higher rate of action-potential firing. In embryonic motoneurons, the fluctuations in peak amplitude of spontaneous PSCs were normally distributed around single peaks with modal values similar to those of mPSCs. These data indicated that early in synapse differentiation spontaneous PSCs were primarily composed of currents generated by quantal release. After birth, mean mPSC amplitude increased by 50% but mean quantal current amplitude did not change. Synchronous, multiquantal release was apparent in postnatal motoneurons only in high-K+ extracellular solution. Comparison of the properties of miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs) demonstrated that mean mEPSC frequency was higher than mIPSC frequency, suggesting that either excitatory synapses outnumbered inhibitory synapses or that the probability of excitatory transmitter release was higher than the release of inhibitory neurotransmitters. The finding that mIPSC duration was several-fold longer than mEPSC duration implied that despite their lower frequency, inhibitory currents could modulate motoneuron synaptic integration by shunting incoming excitatory inputs for prolonged time intervals.     

Fibronectin expression in the developing human spinal cord, nerves, and ganglia

The genetic mechanisms that control asymmetric cell divisions--yielding progeny cells that differ from one another--have been conserved among prokaryotes, eukaryotic microbes, and higher organisms. All use the paradigm of regulatory protein localization as a way of translating genetic information into three-dimensional space.     

Pre- versus postinjury effects of intravenous GABAergic anesthetics on formalin-induced Fos immunoreactivity in the rat spinal cord

We evaluated the suppression of spinal Fos-like immunoreactivity (FLI) by i.v. anesthetics in the rat formalin model. Preformalin injection (1.5% subcutaneously) treatment groups included i.v. saline controls and three i.v. GABAergic anesthetic groups (pentobarbital 20 mg/kg, propofol 10 mg/kg, or alphaxalone 1.5 mg/kg; n = 12 per group). After perfusion 2 h postformalin, spinal cords were dissected, sliced at 30 microm, and processed by immunoperoxidase staining with an antibody against the Fos protein. Quantification and determination of the laminar distribution of Fos-labeled nuclei were performed at the L4-5 spinal level ipsilateral to formalin injection. Drug groups demonstrating FLI suppression were comparatively studied in a 5-min postformalin treatment group. Pentobarbital pretreatment failed to suppress FLI. However, significant reductions (percent decrease) of FLI were observed with propofol (63%) and alphaxalone (30%) compared with saline controls. Pre- versus postformalin comparison studies showed that propofol, but not alphaxalone, suppressed FLI more effectively when given preformalin. Given the observed inconsistencies between this study of Fos expression and our previous behavioral study, it is questionable whether anesthetic modulation of noxious stimulus-induced FLI parallels that of behavioral responses. Implications: In this study, we examined whether i.v. general anesthetics (propofol, alphaxalone, and pentobarbital) prevent injury-induced spinal cord changes. We measured spinal Fos protein after rats received anesthetics before versus after a formalin injection. Fos inhibition patterns were inconsistent with behavioral studies of these anesthetics, suggesting that Fos inhibition does not always correlate with behavioral analgesia.