In vivo imaging of axonal degeneration and regeneration in the injured spinal cord

The poor response of central axons to transection underlies the bleak prognosis following spinal cord injury. Here, we monitor individual fluorescent axons in the spinal cords of living transgenic mice over several days after spinal injury. We find that within 30 min after trauma, axons die back hundreds of micrometers. This acute form of axonal degeneration is similar in mechanism to the more delayed Wallerian degeneration of the disconnected distal axon, but acute degeneration affects the proximal and distal axon ends equally. In vivo imaging further shows that many axons attempt regeneration within 6-24 h after lesion. This growth response, although robust, seems to fail as a result of the inability of axons to navigate in the proper direction. These results suggest that time-lapse imaging of spinal cord injury may provide a powerful analytical tool for assessing the pathogenesis of spinal cord injury and for evaluating therapies that enhance regeneration.     

Phosphate transport after acute changes in total NAD content in renal proximal tubules

Suspensions of proximal tubules were obtained by collagenase digestion of rat renal cortex followed by centrifugation on a percoll gradient. NAD content in tubules incubated at 37 degrees C was decreased by 40-60% compared with tubules incubated at 4 degrees C. This change occurred within 30 min and was maintained for up to 2 hr. Inhibitors of NAD hydrolysing enzymes prevented the depletion of cellular NAD at 37 degrees C. Acute changes in proximal tubule NAD content at 37 degrees C were not accompanied by changes in phosphate uptake by brush border membrane vesicles subsequently prepared from the same tubules. In contrast, incubation of tubules with parathyroid hormone (10(-6) M) produced the expected inhibition (20%) of brush border membrane transport of phosphate. One implication of these findings is that acute changes in total NAD content of proximal tubules at 37 degrees C may not influence the phosphate transport system in the renal brush border membrane. Other interpretations are discussed.     

Altered levels of PGF in cat spinal cord tissue following traumatic injury.

Previous studies by others indicated that PGs were present in brain, spinal cord, and c.s.f. of several mammalian species. In the present study we compared levels of PGE and PGF by R.I.A. in spinal cord tissue from traumatized cats and cats pretreated with indomethacin prior to trauma to those of baseline and sham operated controls in order to assess for the first time, to our knowledge, whether meaningful changes in levels of PGE and PGF could be detected which might shed new light on the etiology of spinal cord trauma. Levels of PGF (nanograms/gram wet wt) in the cord segment immediately adjacent to the point of trauma were 8.05 +/- 1.50, and 13.13 +/- 1.38 for baseline and sham operated cats respectively. Spinal trauma led to more than a 100% increase in PGF levels to 29.26 +/- 3.58. Although pretreatment with indomethacin 30 min prior to trauma gave the expected blockade of the PGF response to trauma, a measurable level of PGF (2.55 +/- 0.17) was found in the cord after indomethacin. Cord levels of PGF declined after 3 hr in both sham operated and traumatized animals. PGF was maximally stimulated by trauma during the first 3 hr with little effect at 72 hr. Although carefully examined, PGE levels in cat spinal cord appeared to be virtually unaffected by trauma. These findings clearly demonstrate for the first time that traumatic injury to the spinal cord is accompanied by marked increases in PG levels at the site of trauma, and that the observed elevation in PGF in response to trauma can be blocked by indomethacin in vivo. Whether PGF changes are causally related to the etiology of spinal cord trauma, or merely represent a manifestation of PG release as a result of non-specific tissue injury, remains to be seen.     

Changes in intra-axonal calcium distribution following nerve crush

We used the oxalate-pyroantimonate method to demonstrate the ultrastructural distribution of calcium within rat sciatic nerve 4 h after a crush injury. In normal nerve there are discrete gradients of axoplasmic calcium precipitate with the amount of precipitate decreasing in the axoplasm beneath the Schmidt Lantermann clefts and in the paranodal regions at the node of Ranvier. Near the crush site a marked increase in endoneurial and intra-axonal calcium precipitate correlated with morphologic evidence of axonal degeneration. More distant from the crush site, both in the distal segment destined to degenerate and in the proximal segment destined to regenerate, the most prominent finding was a loss of the normal gradient of precipitate beneath the Schmidt Lantermann clefts. The calcium influx at the crush site corresponds to the known role of calcium in triggering degeneration. The alterations in the distal axon may be an early stage leading to degeneration. Alteration in calcium distribution in the proximal nerve stump may play a role in the regulation of the response to injury.     

Altered levels of PGF in cat spinal cord tissue following traumatic injury

Previous studies by others indicated that PGs were present in brain, spinal cord, and c.s.f. of several mammalian species. In the present study we compared levels of PGE and PGF by R.I.A. in spinal cord tissue from traumatized cats and cats pretreated with indomethacin prior to trauma to those of baseline and sham operated controls in order to assess for the first time, to our knowledge, whether meaningful changes in levels of PGE and PGF could be detected which might shed new light on the etiology of spinal cord trauma.Levels of PGF (nanograms/gram wet wt) in the cord segment immediately adjacent to the point of trauma were 8.05 ± 1.50, and 13.13 ± 1.38 for baseline and sham operated cats respectively. Spinal trauma led to more than a 100% increase in PGF levels to 29.26 ± 3.58. Although pretreatment with indomethacin 30 min prior to trauma gave the expected blockade of the PGF response to trauma, a measurable level of PGF (2.55 ± 0.17) was found in the cord after indomethacin. Cord levels of PGF declined after 3 hr in both sham operated and traumatized animals. PGF was maximally stimulated by trauma during the first 3 hr with little effect at 72 hr. Although carefully examined, PGE levels in cat spinal cord appeared to be virtually unaffected by trauma.These findings clearly demonstrate for the first time that traumatic injury to the spinal cord is accompanied by marked increases in PG levels at the site of trauma, and that the observed elevation in PGF in response to trauma can be blocked by indomethacin $\text{in vivo}$. Whether PGF changes are causally related to the etiology of spinal cord trauma, or merely represent a manifestation of PG release as a result of non-specific tissue injury, remains to be seen.     

Direct evidence that burst but not sustained secretion of prolactin stimulated by thyrotropin-releasing hormone is dependent on elevation of cytoplasmic calcium

Thyrotropin-releasing hormone stimulation of prolactin secretion from rat pituitary (GH3) cells is biphasic with a secretory burst (0-2 min) at a higher rate, followed by sustained secretion (beyond 2 min) at a lower rate. Based on the effects of calcium ionophores, K+ depolarization, and diacylglycerol (or phorbol esters), it was suggested that the secretory burst is dependent on elevation of cytoplasmic free calcium concentration [( Ca2+]i) whereas sustained secretion is mediated by lipid-activated protein phosphorylation. In this study, we pretreated GH3 cells with 0.03 mM arachidonic acid to abolish thyrotropin-releasing hormone-induced elevation of [Ca2+]i (Kolesnick, R. N., and Gershengorn, M. C. (1985) J. Biol. Chem. 260, 707-713). In control cells, basal secretion was 0.7 +/- 0.2 ng/10(6) cells/min which increased to 8.3 +/- 0.8 between 0 and 2 min after TRH and remained elevated at 3.3 +/- 0.2 between 2-10 min. In cells pretreated with arachidonic acid, TRH stimulated prolactin secretion to only 2.6 +/- 0.3 ng/10(6) cells/min between 0 and 2 min and to 3.2 +/- 0.2 between 2 to 10 min; these values are not different from each other nor from the response between 2 and 10 min in control cells. K+ depolarization, which elevates [Ca2+]i even in arachidonic acid-pretreated cells but does not affect lipid metabolism, caused only a secretory burst. Bovine serum albumin, which binds free arachidonic acid and reverses arachidonic acid inhibition of TRH-induced elevation of [Ca2+]i, reversed the inhibition of the secretory burst stimulated by TRH. These studies present direct evidence that the burst of prolactin secretion stimulated by TRH is dependent on an elevation of [Ca2+]i whereas the sustained phase of secretion is independent of such elevation.     

Ca2+ transients induced by thyrotropin-releasing hormone rapidly lose their ability to cause release of prolactin

To investigate the relationship of changes in cytosolic free calcium concentrations [( Ca2+]c) caused by TRH to changes in PRL secretion, we simultaneously monitored PRL release and [Ca2+]c, using the fluorescent Ca2+ indicator indo-1, in freshly isolated perifused cells from rat anterior pituitary glands. We found that a 30-sec pulse of 100 nM TRH triggered a transient spike of [Ca2+]c, but prolonged PRL release for up to 30 min; continuous administration of TRH caused a sustained elevation in [Ca2+]c, but the same pattern and amount of PRL release as that caused by the pulse of TRH. PRL secretion was refractory to further pulses of TRH given at 10-min intervals for 40 min, but did respond to a second pulse of TRH given 40 min after the first pulse with no intervening pulses. Pulses of TRH given every 10 min still triggered spikes of [Ca2+]c of the same magnitude as the first pulse, indicating that the cause of the refractory state must occur at a post-receptor step that is after the mobilization of [Ca2+]c. A 30-sec pulse of a high concentration of KCl caused a transient spike of [Ca2+]c and transient, not prolonged, release. Additional pulses of KCl cause progressively less PRL release, although the magnitude of the spikes in [Ca2+]c did not change.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of neural cell adhesion molecule in spinal cords following a complete transection

Neural cell adhesion molecule (NCAM) regulates tissue organization during development and in the adult. NCAM upregulation occurs after an injury to brains and sciatic nerves. However, little is known about NCAM expression after spinal cord injury (SCI). By using a complete spinal cord transection with a 5 mm tissue removal, an increase in the NCAM level is detected in spinal cord stumps proximal and distal to the transection site at 1 d and 3 d post injury, while its expression at 8 d is declined to a lower level than that observed in sham-operated spinal cords. The strong NCAM expression is present in motor neurons at 3 d post transection whereas the intensive NCAM immunostaining is localized in dorsal sensory and corticospinal fiber tracts at 8 d following injury. Collectively, NCAM level is elevated and strongly expressed in dorsal fiber tracts after SCI, implying that the endogenous process for spinal cord regeneration may take place after SCI.     

Cardiovascular effect of intravenously administered thyrotropin-releasing hormone and its concentration in push-pull perfusion of the fourth ventricle in conscious and pentobarbital-anesthetized rats

Changes in the concentration of thyrotropin-releasing hormone (TRH) in cerebrospinal fluid (CSF) were examined by the push-pull perfusion method after intravenous (i.v.) administration of the peptide in conscious and pentobarbital-anesthetized rats. The concentration of endogenous TRH in the perfusate was not changed during the 160-min perfusion period and was similar to that in the CSF (0.92 +/- 0.26 ng/ml) collected before the perfusion in conscious as well as in anesthetized rats. After i.v. administration of TRH (5 mg/kg) to the conscious rats, the peptide concentration in the perfusate increased to 42.23 +/- 14.33 ng/ml during the first 20 min and gradually returned to the basal level 2 hr after administration. The total amount of TRH detected in the perfusate was 20.0 ng. It was reduced by 75% in the anesthetized animals. The increases in blood pressure and heart rate, seen after i.v. as well as intracerebroventricular administration of TRH in the conscious rats, was significantly inhibited in the anesthetized rats. These results indicate that systemically administered TRH exerts its cardiovascular effect at central site(s), and that the transportation and the effect of the peptide is suppressed by pentobarbital anesthesia.     

Fluctuation of the plasma TRH level in normal subjects in a 4-hour observation period

Radioimmunoassayable TRH and TSH were measured in plasma samples taken at 5 min intervals for 4 hr (2100-0200 hr) from 4 normal male subjects. Three subjects showed a TSH surge at 2135 hr, 2455 hr and 0150 hr, respectively. The mean plasma TRH level of the 4 subjects was 10.3-11.7 pg/ml. Plasma TRH showed random fluctuation, which did not coincide with the nocturnal increase in plasma TSH.     

Response of thyroid hormones in peripheral and thyroid venous blood to TRH administration in man

Thyroid vein and cubital vein samples were collected simultaneously in 6 moderately hypercalcemic patients and 1 eucalcemic hypothyroid patient, and thyroid hormones were measured in the serum in the basal state, as well as 30 and 60 min after intravenous administration of TRH. No gradient was detectable between peripheral and thyroid blood in the case of T4, and no significant changes were observed following TRH. The levels of T3 were higher in the thyroid venous effluent than in the periphery and a marked increase occurred following TRH. Serum thyroglobulin also increased following TRH, but there was no peripheral vs. thyroid gradient. Calcitonin demonstrated a marked positive gradient in the thyroid vein compared to the periphery, but no change was observed following TRH. It is concluded that the patterns of response of individual thyroid hormones reflect differences in their secretion and, specifically, that intrathyroid conversion of T4 to T3 occurs during the thyroid hormone secretion.     

Spontaneous firing in the isolated anucleate axonal segment of an identified crayfish motoneuron

The aim of this study was to present evidence for prolonged spontaneous firing in an anucleate axonal segment of an identifiable crayfish anal motoneuron L (AML) and to determine the initiation site of this firing. AML has its soma in the 6th abdominal ganglion (A6). By separating a nerve with the AML axon from A6 and the target muscle, various lengths of an anucleate AML axonal segment were procured. Then, AML activity was recorded extracellularly for 14-26 hr from the distal end of this axonal segment. This segment (n=19) exhibited spontaneous firing, which occurred without any stimulation 0.03-5.13 hr after the A6-cut and persisted tonically for 0.20-19.98 hr. During firing, the frequency augmented gradually, whereas the amplitude decreased gradually. There was no significant correlation between latency and duration of the firings. No correlation was noted between latency and length of the axonal segment or its size, or between duration and length or size. These results revealed that the anucleate AML axon itself can inherently generate prolonged firing. The delay in the appearance of AML impulses between the proximal and distal regions at the same axonal segment proved that the firing occurred proximally. There was no significant difference in delays between firing following the A6-cut and the spontaneous firing observed after the A6-cut. This suggests that the initiation site of the spontaneous firing is at the proximal end of the AML axonal segment, since the AML firing following the A6-cut occurs at its cut end.     

人脐带间充质干细胞对脊柱骨折大鼠愈合及神经功能的影响

摘要 目的：探讨人脐带间充质干细胞(Human umbilical cord mesenchymal stem cells,hUC-MSCs)对脊柱骨折大鼠愈合及神经功能的影响。方法：脊柱骨折Sprague-Dawley雄性大鼠模型30只随机分为hUC-MSCs组与对照组,各15只。hUC-MSCs组大鼠在骨折部位移植0.5 mL的hUC-MSCs(细胞浓度为2×10⁶/mL),对照组大鼠移植同体积的生理盐水,记录大鼠愈合及神经功能变化情况。结果：两组造模后15 min、30 min、90 min的平均动脉压都波动明显,不过组间对比差异无统计学意义(P>0.05)。与造模后2 w对比,两组造模后4 w的神经功能BBB评分均升高,且hUC-MSCs组造模后2 w、4 w的神经功能BBB评分都高于对照组(P<0.05)。hUC-MSCs组造模后8 w的骨体积分数高于对照组(P<0.05)。hUC-MSCs组骨折部位附近有少量骨痂生长,骨折线逐渐消失;骨痂已明显包裹骨折部位。hUC-MSCs组造模后8 w的脊髓细胞凋亡指数低于对照组(P<0.05)。结论：hUC-MSCs在脊柱骨折大鼠的应用能促进骨折愈合与改善神经功能,也可以抑制脊髓细胞凋亡,从而发挥很好的治疗作用。     

Pretreatment with calpain inhibitor CEP-4143 inhibits calpain I activation and cytoskeletal degradation, improves neurological function, and enhances axonal survival after traumatic spinal cord injury

The pathophysiology of traumatic spinal cord injury (SCI) involves abnormal activation of the neutral cysteine protease calpain I (EC 3.4.22.17). In the present study we examined the effect of the calpain inhibitor CEP-4143 on cytoskeletal protection and neurological recovery after SCI in adult rats. Microinjection of 50 mM CEP-4143 into the T7 vertebral segment 10 min before a 35-g clip compression injury resulted in inhibition of calpain activation at 2 and 4 h postinjury, as determined by western blotting for calpain I-mediated spectrin degradation, and significantly attenuated the degradation of dephosphorylated NF200 neurofilament protein at 4 and 8 h postinjury. To examine the in vivo chronic neuroprotective effects of CEP-4143, animals underwent microinjection with saline or 50 mM CEP-4143 10 min before injury, followed by weekly blinded behavioral assessments for 6 weeks. Animals receiving CEP-4143 treatment showed significant improvement over saline-treated controls on the Basso Beattie Bresnahan locomotor rating scale (p < 0.02) and inclined plane test (p < 0.05). Counts of neurons in the red nucleus retrogradely labeled by fluoro-gold after introduction distal to the injury site were significantly higher in CEP-4143-treated animals. Finally, morphometric assessment of the injury site by computer-assisted image analysis revealed significant tissue preservation in CEP-4143-treated animals. We conclude that the calpain antagonist CEP-4143 exhibits biochemical, behavioral, and anatomical neuroprotection following traumatic SCI.     

Growth hormone, thyrotropin and prolactin responses to simultaneous administration of human growth hormone-releasing factor and thyrotropin releasing hormone in the bovine

The effects of intravenous injection of synthetic human pancreatic growth hormone-releasing factor-44-NH2 (hpGRF-44) and synthetic thyrotropin releasing hormone (TRH), or hpGRF-44 in combination with TRH on growth hormone (GH), thyrotropin (TSH), and prolactin (PRL) release in dairy female calves (6- and 12-month-old) were studied. When 0.25 microgram of hpGRF-44 per kg of body weight (bw) was injected in combination with TRH (1.0 microgram per kg of bw), the mean plasma GH concentration of the 12-month-old calves rose to a maximum level of 191.5 ng/ml (P less than 0.001) at 15 min from the value of 6.8 ng/ml before injection at 0 min. The maximum level was 3.1 and 6.1 times as high as the peak values obtained after injection of hpGRF-44 (0.25 microgram per kg of bw) and TRH (1.0 microgram per kg of bw), respectively (P less than 0.001). The area under the GH response curve for the 12-month-old calves for 3 hr after injection of hpGRF-44 in combination with TRH was 2.5 times as large as the sum of the areas obtained by hpGRF-44 and TRH injections. In contrast, the mean plasma GH level was unchanged in saline injected calves. The magnitudes of the first and the second plasma GH responses in the 6-month-old calves to two consecutive injections of hpGRF-44 in combination with TRH at a 3-hr interval were very similar. The peak values of plasma GH in the calves after hpGRF-44 injection were 2-4 times as high as those after TRH injection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regional Levels of Lactate and Norepinephrine After Experimental Brain Injury

Abstract: The recently developed controlled cortical impact model of brain injury in rats may be an excellent tool by which to attempt to understand the neurochemical mechanisms mediating the pathophysiology of traumatic brain injury. In this study, rats were subjected to lateral controlled cortical impact brain injury of low grade severity; their brains were frozen in situ at various times after injury to measure regional levels of lactate, high energy phosphates, and norepinephrine. Tissue lactate concentration in the injury site left cortex was increased in injured animals by sixfold at 30 min and twofold at 2.5 h and 24 h after injury ( p < 0.05). At all postinjury times, lactate concentration was also increased in injured animals by about twofold in the cortex and hippocampus adjacent to the injury site ( p < 0.05). No significant changes occurred in the levels of ATP and phosphocreatine in most of the brain regions of injured animals. However, in the primary site of injury (left cortex), phosphocreatine concentration was decreased by 40% in injured animals at 30 min after injury ( p < 0.05). The norepinephrine concentration was decreased in the injury site left cortex of injured animals by 38% at 30 min, 29% at 2.5 h, and 30% at 24 h after injury ( p < 0.05). The level of norepinephrine was also reduced by ∼20% in the cortex adjacent to the injury site in injured animals. The present results suggest that controlled cortical impact brain injury produces disorder in the neuronal oxidative and norepinephrine metabolism.     

Antecedent acute kidney injury worsens subsequent endotoxin-induced lung inflammation in a two-hit mouse model

Acute kidney injury (AKI) contributes greatly to morbidity and mortality in critically ill adults and children. Patients with AKI who subsequently develop lung injury are known to suffer worse outcomes compared with patients with lung injury alone. Isolated experimental kidney ischemia alters distal lung water balance and capillary permeability, but the effects of such an aberration on subsequent lung injury are unknown. We present a clinically relevant two-hit murine model wherein a proximal AKI through bilateral renal ischemia (30 min) is followed by a subsequent acute lung injury (ALI) via intratracheal LPS endotoxin (50 μg at 24 h after surgery). Mice demonstrated AKI by elevation of serum creatinine and renal histopathological damage. Mice with ALI and preexisting AKI had increased lung neutrophilia in bronchoalveolar lavage fluid and by myeloperoxidase activity over Sham-ALI mice. Additionally, lung histopathological damage was greater in ALI mice with preexisting AKI than Sham-ALI mice. There was uniform elevation of monocyte chemoattractant protein-1 in kidney, serum, and lung tissue in animals with both AKI and ALI over those with either injury alone. The additive lung inflammation after ALI with antecedent AKI was abrogated in MCP-1-deficient mice. Taken together, our two-hit model demonstrates that kidney injury may prime the lung for a heightened inflammatory response to subsequent injury and MCP-1 may be involved in this model of kidney-lung cross talk. The model holds clinical relevance for patients at risk of lung injury after ischemic injury to the kidney.     

Effects of thyrotropin releasing hormone on human sudomotor and cutaneous vasomotor activities

At an ambient temperature of 34-41 degrees C (rh = 40%) forearm sweat rates were measured by capacitance hygrometry in 9 male volunteers. Thyrotropin releasing hormone (TRH) was infused intravenously at 0.1 mg.min-1 for 20 to 30 min. Sweat rate increased rapidly within a minute after initiation of TRH infusion, decreased rapidly after the peak sweat rate was attained in 2-5 min of TRH infusion, and then levelled off in 6-10 min near the level before TRH infusion. Core temperature (Tre, Tty) started to decline at the time of the peak sweat rate and levelled off almost coincidentally with the levelling off in sweat rate. Average values for the rate of sweat expulsions (Fsw), sweat rate and mean body temperature (Tb) were obtained from the data of the last 10 min period of TRH infusion. The regression line for the relationship of Fsw to Tb shifted during the TRH infusion to the left of the line for the control; that of sweat rate to Fsw hardly shifted. At an ambient temperature of 24-27 degrees C TRH produced vasodilation as evidenced by an increase in skin blood flow (measured by means of thermal distribution), an increase in amplitude of the photoelectric plethysmogram and an elevation of skin temperature in the finger tips. It is suggested that TRH may act, either directly or indirectly, on the central thermoregulatory mechanism (or on the thermoreceptive mechanism) to lower the reference temperature for heat dissipation.