Cannabidiol reduces lesion volume and restores vestibulomotor and cognitive function following moderately severe traumatic brain injury.

Despite the high incidence of traumatic brain injury (TBI), there is no universal treatment to safely treat patients. Blunt brain injuries destroy primary neural tissue that results in impaired perfusion, excessive release of glutamate, inflammation, excitotoxicity, and progressive secondary neuronal cell death. We hypothesized that administration of cannabidiol (CBD) directly to a brain contusion site, will optimize delivery to the injured tissue which will reduce local neural excitation and inflammation to spare neural tissue and improve neurological outcome following TBI. CBD was infused into a gelfoam matrix forming an implant (CBDi), then applied over the dura at the contusion site as well as delivered systemically by injection (CBD.IP). Post-injury administration of CBDi+IP greatly reduced defecation scores, lesion volume, the loss of neurons in the ipsilateral hippocampus, the number of injured neurons of the contralateral hippocampus, and reversed TBI-induced glial fibrillary acidic protein (GFAP) upregulation which was superior to either CBD.IP or CBDi treatment alone. Vestibulomotor performance on the beam-balance test was restored by 12 days post-TBI and sustained through 28 days. CBDi+IP treated rats exhibited preinjury levels of spontaneous alternation on the spontaneous alternation T-maze. In the object recognition test, they had greater mobility and exploration of novel objects compared to contusion or implant alone consistent with reduced anxiety and restored cognitive function. These results suggest that dual therapy by targeting the site of injury internally with a CBD-infused medical carrier followed by systemic supplementation may offer a more effective countermeasure than systemic or implant treatment alone for the deleterious effects of penetrating head wounds.

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 alpha7 nicotinic acetylcholine receptors in activation of tyrosine hydroxylase and dopamine beta-hydroxylase gene expression in PC12 cells.

Nicotine treatment increases intracellular free Ca(2+) concentration [Ca(2+)](i), stimulates catecholamine release, and elevates gene expression for the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH). However, the type of nicotinic acetylcholine receptors (nAChRs) mediating these events is unclear. The nAChR receptor antagonists alpha-bungarotoxin (alphaBTX) and methyllycaconitine greatly reduced the nicotine-triggered initial transient rise in [Ca(2+)](i) and prevented the second prolonged elevation of [Ca(2+)](i), suggesting the involvement of alpha7 nAChRs. Two specific alpha7 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 [Ca(2+)](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 [Ca(2+)](i) for up to 6 h. The increase in [Ca(2+)](i) or the elevation in TH mRNA by 3-CA was completely inhibited by alphaBTX. This study, for the first time, implicates stimulation of alpha7 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 beta(2)-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 beta(2)-adrenoceptor activation. Optimization of beta(2)-agonist treatment of muscular dystrophy in mdx mice may lead to a useful therapeutic modality for human forms of the disease.

Clenbuterol, a beta(2)-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.

Differing temporal roles of Ca2+ and cAMP in nicotine-elicited elevation of tyrosine hydroxylase mRNA.

The involvement of cAMP- and Ca2+-mediated pathways in the activation of tyrosine hydroxylase (TH) gene expression by nicotine was examined in PC-12 cells. Extracellular Ca2+ and elevations in intracellular Ca2+ concentration ([Ca2+]i) were required for nicotine to increase TH mRNA. The nicotine-elicited rapid rise in [Ca2+]i was inhibited by blockers of either L-type or N-type, and to a lesser extent P/Q-, but not T-type, voltage-gated Ca2+ channels. With continual nicotine treatment, [Ca2+]i returned to basal levels within 3-4 min. After a lag of approximately 5-10 min, there was a smaller elevation in [Ca2+]i that persisted for 6 h and displayed different responsiveness to Ca2+ channel blockers. This second phase of elevated [Ca2+]i was blocked by an inhibitor of store-operated Ca2+ channels, consistent with the observed generation of inositol trisphosphate. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM), when added before or 2 h after nicotine, prevented elevation of TH mRNA. Nicotine treatment significantly raised cAMP levels. Addition of the adenylyl cyclase inhibitor 2', 5'-dideoxyadenosine (DDA) prevented the nicotine-elicited phosphorylation of cAMP response element binding protein. DDA also blocked the elevation of TH mRNA only when added after the initial transient rise in [Ca2+]i and not after 1 h. This study reveals that several temporal phases are involved in the induction of TH gene expression by nicotine, each of them with differing requirements for Ca2+ and cAMP.

Clenbuterol, a beta2-adrenoceptor agonist, reduces scoliosis due to partial transection of rat spinal cord.

Injury to the spinal cord often results in abnormal lateral curvature of the spine, or scoliosis, that is associated with neuromuscular weakness. The lateral curvature of the spine is thought to be a consequence of insufficient or asymmetrical loading of the vertebrae. To study neuromuscular scoliosis, an animal model of spinal cord injury was used in which the spinal cord was partially (3/4) transected, with the left lateral columns left intact. Partial transection of the spinal cord in the rat caused scoliosis that was maximal four to five vertebrae distal to the lesion site. As in previous experiments involving unilateral spinal cord lesions, the scoliotic curves were convex on the weakened side. Subtotal transection at T5 or T11 resulted in lateral displacement of vertebrae T9-T12 or L2-L5, respectively, of up to 11 mm. Interestingly, this vertebral displacement is greatly reduced by clenbuterol, a beta2-adrenoceptor agonist that has been found to retard loss of muscle contractility and bone mineralization due to denervation. Together these results suggest that stimulation of beta2-receptors opposes vertebral unloading due to neuromuscular weakness and thereby acts as a countermeasure to scoliosis.

Clenbuterol, a beta 2-agonist, retards wasting and loss of contractility in irradiated dystrophic mdx muscle.

Treatment with the adrenergic beta 2-receptor agonist clenbuterol prevented, in dystrophic muscle from mdx mice, a pronounced loss of contractile strength that is observed after blockade of muscle regeneration with gamma irradiation. In addition, muscle mass and myosin content were greater (62-109%) in irradiated hindlimbs from clenbuterol-treated mdx mice, whereas the effects of the beta 2-agonist were relatively smaller (12-21%) in the nonirradiated hindlimbs. Together, these results suggest that beta 2-agonists can antagonize degenerative processes occurring in muscle fibers lacking dystrophin.

Protein phosphatase inhibitors prolong Ca(2+)-transients and divalent cation-dependent action potentials in leech Retzius cells.

Elevation of [K+]o for 30 s from 4 to 120 mM produced a fast and reversible depolarization and transient increase in [Ca2+]i in fura-2 loaded Retzius cells of the leech. The protein phosphatase inhibitor, okadaic acid, significantly slowed the return of [Ca2+]i toward baseline without affecting the amplitude of depolarization or rate of repolarization. Furthermore, okadaic acid and another phosphatase inhibitor, calyculin A, prolonged Ba(2+)-dependent action potentials. These results suggest that the kinetics of Ca2+ influx may be regulated by the activity of phosphatases PP-1 and/or PP-2A.

Clenbuterol, a beta 2-receptor agonist, reduces net bone loss in denervated hindlimbs.

Clenbuterol treatment for several weeks prevented up to one-third of the reduction in mineralization of femurs and tibias caused by sectioning of the sciatic nerve in young rats. The normalizing effect of clenbuterol on bone mineral content was directly proportional to similar alterations in muscle mass, which in turn could be abolished by ablation of the triceps surae or hindlimb unweighting and reduced by hypophysectomy. In contrast to the effects of inactivity, ovariectomy caused small reductions (2-4%) in bone density that were not affected by clenbuterol and were not accompanied by changes in ash weight. Together, our results suggest that the ability of beta 2-agonists to retard the loss in net muscle mass and enhance contractile tension can oppose net bone loss caused by denervation. Increases in contractile tension caused by beta 2-agonists may enhance the utility of exercise or electrical stimulation as countermeasures for the effects of scoliosis, prolonged bed rest, spinal cord injury, or weightlessness in space on bone mass.

Slow to fast alterations in skeletal muscle fibers caused by clenbuterol, a beta 2-receptor agonist.

Chronic treatment of rats with clenbuterol, a beta 2-receptor agonist (8-12 wk), caused hypertrophy of histochemically identified fast- but not slow-twitch fibers within the soleus, while the mean areas of both fiber types were increased in the extensor digitorum longus (EDL). In contrast, treatment with the beta 2-receptor antagonist, butoxamine, reduced fast-twitch fiber size in both muscles. In the solei and to a lesser extent in the EDLs, the ratio of the number of fast- to slow-twitch fibers was increased by clenbuterol, while the opposite was observed with butoxamine. The muscle fiber hypertrophy observed in the EDL was accompanied by parallel increases in maximal tetanic tension and muscle cross-sectional area, while in the solei, progressive increases in rates of force development and relaxation toward values typical of fast-twitch muscles were also observed. Our results suggest a role of beta 2-receptors in regulating muscle fiber type composition as well as growth.

Clenbuterol, a beta 2-agonist, retards atrophy in denervated muscles.

Denervated soleus, anterior tibialis, and gastrocnemius muscles, but not the extensor digitorum longus, contained 95-110% more protein after 2-3 wk of treatment with the adrenergic beta 2-receptor agonist, clenbuterol, than denervated controls. In addition, the twofold difference in the protein content of denervated solei was paralleled by similar changes in contractile strength and muscle fiber cross-sectional area. In contrast, when the innervated contralateral muscles were examined, the extensor digitorum longus and anterior tibialis showed relatively small increases in protein of 32 and 19%, respectively, whereas the soleus and gastrocnemius were unaffected. The magnitude of the effects of clenbuterol in sparing the mass and functional capacity of denervated muscle suggests that this agent may be important in studies of neuromuscular diseases and disuse atrophy.

Regulation of Ca2+-dependent protein turnover in skeletal muscle by thyroxine.

Dantrolene, an agent that inhibits Ca2+ mobilization, improved protein balance in skeletal muscle, as thyroid status was increased, by altering rates of protein synthesis and degradation. Thyroxine (T4) caused increases in protein degradation that were blocked by leupeptin, a proteinase inhibitor previously shown to inhibit Ca2+-dependent non-lysosomal proteolysis in these muscles. In addition, T4 abolished sensitivity to the lysosomotropic agent methylamine and the autophagy inhibitor 3-methyladenine, suggesting that T4 inhibits autophagic/lysosomal proteolysis.

Regulation of protein degradation in muscle by calcium. Evidence for enhanced nonlysosomal proteolysis associated with elevated cytosolic calcium.

Calcium-dependent regulation of intracellular protein degradation was studied in isolated rat skeletal muscles incubated in vitro in the presence of a large variety of agents known to affect calcium movement and distribution. A23187, KC1, sucrose, and 8-(diethylamino)octyl-3,4, 5-trimethoxybenzoate hydrochloride increase proteolysis while tetracaine, verapamil, and low extracellular calcium caused significant decreases. Additionally, dantrolene decreases proteolysis in the presence of depolarizing levels of potassium, while it has no effect on degradation in normal media. The dose dependence of calcium ionophore A23187 on proteolysis and contracture tension are parallel. Furthermore, excess KC1 and hypertonic solutions increased protein degradation at doses reported to cause tension. Thus, the parallel increase in proteolysis and tension in response to various agents supports the hypothesis that protein degradation in muscle is regulated by calcium. To determine the responsible proteolytic systems involved in calcium-dependent degradation, the effect of different classes of protease inhibitors was tested. Addition of the inhibitors leupeptin and E-64-c blocked the A23187-induced increase in degradation. Since proteases sensitive to these agents are present in both the sarcoplasm and lysosomes, known lysosomotropic agents, methylamine and chloroquine, as well as 3-methyladenine, a specific autophagy inhibitor, were used in combination with A23187. These agents did not inhibit calcium ionophore-induced proteolysis, although these three agents selectively inhibited enhanced degradation seen in the absence of insulin, demonstrating an autophagic/lysosomal pathway in these muscles. Thus, our results suggest that nonlysosomal leupeptin- and E-64-c-sensitive proteases are responsible for calcium-dependent proteolysis in muscle.