Richard J. Zeman

Involvement of α7 Nicotinic Acetylcholine Receptors in Activation of Tyrosine Hydroxylase and Dopamine β‐Hydroxylase Gene Expression in PC12 Cells

Abstract: Nicotine treatment increases intracellular free Ca2+ concentration [Ca2+]i, stimulates catecholamine release, and elevates gene expression for the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH) and dopamine β‐hydroxylase (DBH). However, the type of nicotinic acetylcholine receptors (nAChRs) mediating these events is unclear. The nAChR receptor antagonists α‐bungarotoxin (αBTX) and methyllycaconitine greatly reduced the nicotine‐triggered initial transient rise in [Ca2+]i and prevented the second prolonged elevation of [Ca2+]i, suggesting the involvement of α7 nAChRs. Two specific α7 nicotinic agonists, 3‐(2,4‐dimethoxybenzilidene)anabaseine (DMXB) and E,E‐3‐(cinnamylidene)anabaseine (3‐CA), were found to elicit a small, delayed increase in [Ca2+]i with kinetics and magnitude similar to the second elevation observed with nicotine. This increase was inhibited by the inositol trisphosphate receptor antagonist xestospongin C. Exposure to 3‐CA or DMXB for 6 or 24 h elevated TH and DBH mRNA levels two‐ to fourfold over control levels. These agonists were more effective than nicotine alone in increasing TH and DBH gene expression and significantly elevated [Ca2+]i for up to 6 h. The increase in [Ca2+]i or the elevation in TH mRNA by 3‐CA was completely inhibited by αBTX. This study, for the first time, implicates stimulation of α7 nAChRs in the activation of TH and DBH gene expression.

Clenbuterol reduces degeneration of exercised or aged dystrophic (mdx) muscle

Evidence of dystrophic muscle degeneration in the hind limb muscles of young (20‐week‐old) treadmill‐exercised or aged (87‐week‐old) sedentary mdx mice was greatly reduced by treatment with clenbuterol, a β2‐adrenoceptor agonist. Daily treadmill exercise for 10 weeks increased the size of regions within the mdx plantaris but not the soleus or gastrocnemius muscles, in which necrotic muscle fibers or the absence of fibers was observed. Clenbuterol reduced the size of these abnormal regions from 21% of total muscle cross‐sectional area to levels (4%) found in sedentary mdx mice. In addition, the muscles obtained from aged clenbuterol‐treated mdx or wild‐type mice did not display the extensive fibrosis or fiber loss observed in untreated mdx mice. These observations are consistent with a mechanism of dystrophic muscle degeneration caused by work load–induced injury that is cumulative with aging and is opposed by β2‐adrenoceptor activation. Optimization of β2‐agonist treatment of muscular dystrophy in mdx mice may lead to a useful therapeutic modality for human forms of the disease.

Clenbuterol, a β2-Adrenoceptor Agonist, Improves Locomotor and Histological Outcomes after Spinal Cord Contusion in Rats

An important goal of rehabilitation following spinal cord injury is recovery of locomotor function and muscular strength. In the present studies, we determined whether the beta(2)-agonist, clenbuterol, can improve recovery of locomotor function following spinal cord injury. A model of spinal cord injury was examined in which four graded levels of contusion injury were produced in rats at the level of T10 with a weight-drop device. Locomotor recovery was determined with the Basso, Beattie, and Bresnahan (BBB) scale, which distinguishes between 22 progressive levels of recovery. As observed previously, recovery during the 6 weeks following injury was inversely related to the severity of injury. However, clenbuterol caused substantial enhancement of recovery of locomotor function at the two most severe levels of injury (BBB scores 10-12 vs 2-4). In addition, the extent of recovery was directly related to sparing of spinal cord tissue at the contusion center in both untreated and clenbuterol-treated spinal cords. Optimization of beta(2)-agonist treatment may lead to a useful therapeutic modality for treatment of spinal cord contusion injury.

Clenbuterol retards loss of motor function in motor neuron degeneration mice

Motor neuron degeneration (mnd) mice exhibit lysosomal accumulation of lipofuscin-like material that is associated with progressive loss of motor function and strength. Motor dysfunction scores at 8.5-9 months of age were highly correlated with the occurrence of abnormal spinal motor neurons with eccentric nuclei, although the total numbers of motor neurons were not significantly reduced. Nuclear eccentricity is a characteristic of the axon reaction that results from injury and subsequent compensatory axonal sprouting indicating axonal/synaptic dysfunction in mnd motor neurons. Treatment with clenbuterol, a beta(2)-adrenoceptor agonist that can enhance regeneration of motor neuron axons, opposed the development of motor deficits in parallel with a reduced proportion of motor neurons with eccentric nuclei consistent with improved synaptic function. Clenbuterol also opposed decreases in grip strength and muscle mass suggesting beta(2)-agonist treatment as a potential therapeutic modality for lipofuscinoses.

X-irradiation of the contusion site improves locomotor and histological outcomes in spinal cord-injured rats

We have determined whether X-irradiation of the injury site can oppose tissue loss and improve recovery of locomotor function following contusion injury of the spinal cord. Contusion injury was produced in rats at the level of T10 with a weight drop device. Localized X-irradiation (20 Gy) of the injury site was performed at 20 min and 1, 2, 4, 7, and 17 days postinjury. Locomotor recovery was then determined with the 21-point Basso, Beattie, and Bresnahan (BBB) scale. X-irradiation enhanced recovery of locomotor function during a subsequent 6-week observation period when administered 20 min and 1 or 2 days following contusion injury (final BBB score approximately 7-8). X-irradiation at 4-17 days postinjury did not significantly affect final locomotor scores compared with unirradiated rats (final BBB score approximately 2), in marked contrast to previous studies where X-irradiation applied only at 17-18 days benefitted transection injury. The extent of recovery was directly related to measurements of sparing of spinal cord tissue at the contusion center. Because the treatment time window occurred earlier in contusion than reported for transection injury, the results suggest that contusion injury rapidly initiates underlying radiation-sensitive processes that occur only following a delay of several weeks after transection injury. Further optimization of X-ray treatment may lead to a useful therapeutic modality for use in spinal cord contusion injury.

Involvement of intracellular or extracellular calcium in activation of tyrosine hydroxylase gene expression in PC12 cells

Abstract: The relationship between elevations in intracellular free Ca2+ concentration ([Ca2+]i) by different mechanisms and tyrosine hydroxylase (TH) gene expression was examined. Depolarization by an elevated K+ concentration triggered rapid and sustained increases in [Ca2+]i from a basal level of ∼50 to 110–150 nM and three‐ to fourfold elevations in TH mRNA levels, requiring extracellular calcium but not inositol 1,4,5‐trisphosphate (IP3). On the other hand, bradykinin or thapsigargin, both of which induce release of intracellular calcium stores via IP3 or inhibition of Ca2+‐ATPase, rapidly elevated [Ca2+]i to >200 nM and increased TH gene expression (three‐to fivefold). Confocal imaging showed that the elevations in [Ca2+]i in each case occurred throughout the cyto‐ and nucleoplasm. The initial rise in [Ca2+]i due to either bradykinin or thapsigargin, which did not require extracellular calcium, was sufficient to initiate the events leading to increased TH expression. Consistent with this, the effects of bradykinin on TH expression were inhibited by 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid or 3,4,5‐trimethoxybenzoic acid 8‐(diethylamino)‐octyl ester which chelates or inhibits the release of intracellular calcium, respectively. Bradykinin required a rise in [Ca2+]i for <10 min, as opposed to 10–30 min for depolarization to increase TH mRNA levels. These results demonstrate that although each of these treatments increased TH gene expression by raising [Ca2+]i, there are important differences among them in terms of the magnitude of elevated [Ca2+]i, requirements for extracellular calcium or release of intracellular calcium stores, and duration of elevated [Ca2+]i, indicating the involvement of different calcium signaling pathways leading to regulation of TH gene expression.

Stem Cell Therapy and Curcumin Synergistically Enhance Recovery from Spinal Cord Injury

Acute traumatic spinal cord injury (SCI) is marked by the enhanced production of local cytokines and pro-inflammatory substances that induce gliosis and prevent reinnervation. The transplantation of stem cells is a promising treatment strategy for SCI. In order to facilitate functional recovery, we employed stem cell therapy alone or in combination with curcumin, a naturally-occurring anti-inflammatory component of turmeric (Curcuma longa), which potently inhibits NF-κB. Spinal cord contusion following laminectomy (T9–10) was performed using a weight drop apparatus (10 g over a 12.5 or 25 mm distance, representing moderate or severe SCI, respectively) in Sprague-Dawley rats. Neural stem cells (NSC) were isolated from subventricular zone (SVZ) and transplanted at the site of injury with or without curcumin treatment. Functional recovery was assessed by BBB score and body weight gain measured up to 6 weeks following SCI. At the conclusion of the study, the mass of soleus muscle was correlated with BBB score and body weight. Stem cell therapy improved recovery from moderate SCI, however, it had a limited effect on recovery after severe SCI. Curcumin stimulated NSC proliferation in vitro, and in combination with stem cell therapy, induced profound recovery from severe SCI as evidenced by improved functional locomotor recovery, increased body weight, and soleus muscle mass. These findings demonstrate that curcumin in conjunction with stem cell therapy synergistically improves recovery from severe SCI. Furthermore, our results indicate that the effect of curcumin extends beyond its known anti-inflammatory properties to the regulation of stem cell proliferation.

Recovery from spinal cord injury using naturally occurring antiinflammatory compound curcumin: laboratory investigation.

OBJECT Spinal cord injury (SCI) is a debilitating disease. Primary SCI results from direct injury to the spinal cord, whereas secondary injury is a side effect from subsequent edema and ischemia followed by activation of proinflammatory cytokines. These cytokines activate the prosurvival molecule nuclear factor-κB and generate obstacles in spinal cord reinnervation due to gliosis. Curcumin longa is an active compound found in turmeric, which acts as an antiinflammatory agent primarily by inhibiting nuclear factor-κB. Here, the authors study the effect of curcumin on SCI recovery. METHODS Fourteen female Sprague-Dawley rats underwent T9-10 laminectomy and spinal cord contusion using a weight-drop apparatus. Within 30 minutes after contusion and weekly thereafter, curcumin (60 mg/kg/ml body weight in dimethyl sulfoxide) or dimethyl sulfoxide (1 ml/kg body weight) was administered via percutaneous epidural injection at the injury site. Spinal cord injury recovery was assessed weekly by scoring hindlimb motor function. Animals were killed 6 weeks postcontusion for histopathological analysis of spinal cords and soleus muscle weight evaluation. RESULTS Curcumin-treated rats had improved motor function compared with controls starting from Week 1. Body weight gain significantly improved, correlating with improved Basso-Beattie-Bresnahan scores. Soleus muscle weight was greater in curcumin-treated rats than controls. Histopathological analysis validated these results with increased neural element mass with less gliosis at the contusion site in curcumin-treated rats than controls. CONCLUSIONS Epidural administration of curcumin resulted in improved recovery from SCI. This occurred with no adverse effects noted in experimental animals. Therefore, curcumin treatment may translate into a novel therapy for humans with SCI.

Improved functional recovery with oxandrolone after spinal cord injury in rats.

At present, only the corticosteroid, methylprednisolone, is used for acute spinal cord injury to improve function. However, improvements are modest, and are associated with myopathy and immunosuppression so that alternative treatments are needed. Oxandrolone is an androgenic steroid with potential neuroprotective properties that is used to prevent muscle loss and is not immunosuppressive. Oxandrolone increased locomotor recovery concomitant with reduced loss of cord tissue in a standard weight drop model of spinal cord contusion injury indicating oxandrolone as a possible alternative to methylprednisolone. Oxandrolone also increased axonal sprouting within the ventral horns distal to the injury consistent with formation of relay circuits mediating locomotor recovery.

STEREOTACTIC RADIOSURGERY IMPROVES LOCOMOTOR RECOVERY AFTER SPINAL CORD INJURY IN RATS

OBJECTIVECurrently, because of the precision of stereotactic radiosurgery, radiation can now be delivered by techniques that shape the radiation beam to the tissue target for a variety of clinical applications. This avoids unnecessary and potentially damaging irradiation of surrounding tissues inherent in conventional irradiation, so that irradiation of the minimum volume of tissue necessary for optimal therapeutic benefit can be achieved. Although conventional x-irradiation has been shown to improve recovery from spinal cord injury in animals, the efficacy of targeted irradiation of the injured spinal cord has not been demonstrated previously. The purpose of these studies was to determine whether stereotactic x-irradiation of the injured spinal cord can enhance locomotor function and spare spinal cord tissue after contusion injury in a standard experimental model of spinal cord injury. METHODSContusion injury was produced in rats at the level of T10 with a weight-drop device, and doses of x-irradiation were delivered 2 hours after injury via a Novalis, 6-MeV linear accelerator shaped beam radiosurgery system (BrainLAB USA, Westchester, IL) in 4 sequential fractions, with beam angles 60 to 70 degrees apart, at a rate of 6.4 Gy/minute. The target volume was a 4 × 15-mm cylinder along the axis of the spinal cord, with the isocenter positioned at the contusion epicenter. Locomotor function was determined for 6 weeks after injury with the 21-point Basso, Beattie, and Bresnahan (BBB) locomotor scale and tissue sparing in histological sections of the spinal cord. RESULTSLocomotor function recovered progressively during the 6-week postinjury observation period. BBB scores were significantly greater in the 10-Gy x-irradiated group compared with controls (9.4 versus 7.3; P < 0.05), indicating hind limb weight support or dorsal stepping in contrast to hind limb joint mobility without weight bearing. Doses in the range of 2 to 10 Gy increased BBB scores progressively, whereas greater doses of 15 to 25 Gy were associated with lower BBB scores. The extent of locomotor recovery after treatment with x-irradiation correlated with measurements of spared spinal cord tissue at the contusion epicenter. CONCLUSIONThese results suggest a beneficial role for stereotactic radiosurgery in a rat model of acute spinal cord contusion injury and raise hopes for human treatment strategies. Additional animal studies are needed to further define potential benefits.