Altered monoaminergic levels, spasticity, and balance disability following repetitive blast-induced traumatic brain injury in rats.

Spasticity and balance disability are major complications following traumatic brain injury (TBI). Although monoaminergic inputs provide critical adaptive neuromodulations to the motor system, data are not available regarding the levels of monoamines in the brain regions related to motor functions following repetitive blast TBI (bTBI). The objective of this study was to determine if mild, repetitive bTBI results in spasticity/balance deficits and if these are correlated with altered levels of norepinephrine, dopamine, and serotonin in the brain regions related to the motor system. Repetitive bTBI was induced by a blast overpressure wave in male rats on days 1, 4, and 7. Following bTBI, physiological/behavioral tests were conducted and tissues in the central motor system (i.e., motor cortex, locus coeruleus, vestibular nuclei, and lumbar spinal cord) were collected for electrochemical detection of norepinephrine, dopamine, and serotonin by high-performance liquid chromatography. The results showed that norepinephrine was significantly increased in the locus coeruleus and decreased in the vestibular nuclei, while dopamine was significantly decreased in the vestibular nuclei. On the other hand, serotonin was significantly increased in the motor cortex and the lumbar spinal cord. Because these monoamines play important roles in regulating the excitability of neurons, these results suggest that mild, repetitive bTBI-induced dysregulation of monoaminergic inputs in the central motor system could contribute to spasticity and balance disability. This is the first study to report altered levels of multiple monoamines in the central motor system following acute mild, repetitive bTBI.

Effect of Simultaneous Combined Treadmill Training and Magnetic Stimulation on Spasticity and Gait Impairments after Cervical Spinal Cord Injury.

Cervical spinal cord injury (CSCI) can induce lifelong disabilities, including spasticity and gait impairments. The objective of this pre-clinical study was to evaluate the therapeutic effects of simultaneous and combined early locomotor treadmill training (Tm) and injury site magnetic stimulation (TMSsc) on spasticity and gait impairments in a rat model of C6/7 moderate contusion SCI. The Tm training was initiated at post-injury (PI) day 8, whereas TMS treatment was added to Tm 14 days PI, and then the combined therapy (TMSTm) was continued for six weeks. Untreated CSCI animals revealed significant and enduring hindlimb spasticity (measured as velocity-dependent ankle torques and time-locked triceps surae electromyography), significant alterations in limb coordination, and significant reductions in forelimb grip strength. The TMSTm showed significantly lower spasticity, significantly more normal limb coordination (quantitated using three-dimensional (3D) kinematics and Catwalk gait analyses), and significantly greater forelimb grip strength compared with the CSCI untreated controls. In addition, three-dimensional gradient echo and diffusion tensor magnetic resonance imaging showed that TMSTm treated animals had smaller cavity volumes and better preservation of the white matter. In addition, compared with the CSCI untreated animals, the lumbar spinal cord (SC) of the treatment group revealed significant up-regulation of dopamine beta-hydroxylase, glutamic acid decarboxylase, gamma-aminobutyric acid receptor B, and brain-derived neurotrophic factor. The treatment-induced up-regulation of these molecules may have enhanced the activity-induced adaptive plasticity in the SC and contributed to normalization of pre- and post-synaptic reflex regulatory processes. In addition, the TMSTm therapy may have decreased injury-induced progressive maladaptive segmental and descending plasticity. Our data are the first to suggest that an early simultaneous combination of Tm and injury-site TMSsc application can be an effective therapy for CSCI-induced spasticity and gait impairments. These pre-clinical data demonstrated the feasibility and efficacy of a novel therapeutic strategy for SCI-induced spasticity and gait impairments.

Mild closed head traumatic brain injury-induced changes in monoamine neurotransmitters in the trigeminal subnuclei of a rat model: mechanisms underlying orofacial allodynias and headache.

Our recent findings have demonstrated that rodent models of closed head traumatic brain injury exhibit comprehensive evidence of progressive and enduring orofacial allodynias, a hypersensitive pain response induced by non-painful stimulation. These allodynias, tested using thermal hyperalgesia, correlated with changes in several known pain signaling receptors and molecules along the trigeminal pain pathway, especially in the trigeminal nucleus caudalis. This study focused to extend our previous work to investigate the changes in monoamine neurotransmitter immunoreactivity changes in spinal trigeminal nucleus oralis, pars interpolaris and nucleus tractus solitaries following mild to moderate closed head traumatic brain injury, which are related to tactile allodynia, touch-pressure sensitivity, and visceral pain. Our results exhibited significant alterations in the excitatory monoamine, serotonin, in spinal trigeminal nucleus oralis and pars interpolaris which usually modulate tactile and mechanical sensitivity in addition to the thermal sensitivity. Moreover, we also detected a robust alteration in the expression of serotonin, and inhibitory molecule norepinephrine in the nucleus tractus solitaries, which might indicate the possibility of an alteration in visceral pain, and existence of other morbidities related to solitary nucleus dysfunction in this rodent model of mild to moderate closed head traumatic brain injury. Collectively, widespread changes in monoamine neurotransmitter may be related to orofacial allodynhias and headache after traumatic brain injury.

Mild and Mild-to-Moderate Traumatic Brain Injury-Induced Significant Progressive and Enduring Multiple Comorbidities.

Traumatic brain injury (TBI) can produce life-long disabilities, including anxiety, cognitive, balance, and motor deficits. The experimental model of closed head TBI (cTBI) induced by weight drop/impact acceleration is known to produce hallmark TBI injuries. However, comprehensive long-term characterization of comorbidities induced by graded mild-to- mild/moderate intensities using this experimental cTBI model has not been reported. The present study used two intensities of weight drop (1.0 m and 1.25 m/450 g) to produce cTBI in a rat model to investigate initial and long-term disability of four comorbidities: anxiety, cognitive, vestibulomotor, and spinal reflex that related to spasticity. TBI and sham injuries were produced under general anesthesia. Time for righting recoveries post-TBI recorded to estimate duration of unconsciousness, revealed that the TBI mild/moderate group required a mean of 1 min 27 sec longer than the values observed for noninjured sham animals. Screening magnetic resonance imaging images revealed no anatomical changes, mid-line shifts, or hemorrhagic volumes. However, compared to sham injuries, significant long-term anxiety, cognitive, balance, and physiological changes in motor reflex related to spasticity were observed post-TBI for both TBI intensities. The longitudinal trajectory of anxiety and balance disabilities tested at 2, 4, 8, and 18 weeks revealed progressively worsening disabilities. In general, disability magnitudes were proportional to injury intensity for three of the four measures. A natural hypothesis would pose that all disabilities would increase incrementally relative to injury severity. Surprisingly, anxiety disability progressed over time to be greater in the mildest injury. Collectively, translational implications of these observations suggest that patients with mild TBI should be evaluated longitudinally at multiple time points, and that anxiety disorder could potentially have a particularly low threshold for appearance and progressively worsen post-injury.

Closed-Head TBI Model of Multiple Morbidity.

Successful therapy for TBI disabilities awaits refinement in the understanding of TBI neurobiology, quantitative measurement of treatment-induced incremental changes in recovery trajectories, and effective translation to human TBI using quantitative methods and protocols that were effective to monitor recovery in preclinical models. Details of the specific neurobiology that underlies these injuries and effective quantitation of treatment-induced changes are beginning to emerge utilizing a variety of preclinical and clinical models (for reviews see (Morales et al., Neuroscience 136:971-989, 2005; Fujimoto et al., Neurosci Biobehav Rev 28:365-378, 2004; Cernak, NeuroRx 2:410-422, 2005; Smith et al., J Neurotrauma 22:1485-1502, 2005; Bose et al., J Neurotrauma 30:1177-1191, 2013; Xiong et al., Nat Rev Neurosci 14:128-142, 2013; Xiong et al., Expert Opin Emerg Drugs 14:67-84, 2009; Johnson et al., Handb Clin Neurol 127:115-128, 2015; Bose et al., Brain neurotrauma: molecular, neuropsychological, and rehabilitation aspects, CRC Press/Taylor & Francis, Boca Raton, 2015)). Preclinical models of TBI, essential for the efficient study of TBI neurobiology, benefit from the setting of controlled injury and optimal opportunities for biometric quantitation of injury and treatment-induced changes in the trajectories of disability. Several preclinical models are currently used, and each offer opportunities for study of different aspects of TBI primary and secondary injuries (for review see (Morales et al., Neuroscience 136:971-989, 2005; Xiong et al., Nat Rev Neurosci 14:128-142, 2013; Xiong et al., Expert Opin Emerg Drugs 14:67-84, 2009; Johnson et al., Handb Clin Neurol 127:115-128, 2015; Dixon et al., J Neurotrauma 5:91-104, 1988)). The closed-head, impact-acceleration model of TBI designed by Marmarou et al., 1994 (J Neurosurg 80:291-300, 1994), when used to produce mild to moderate TBI, produces diffuse axonal injuries without significant additional focal injuries of the brain (Morales et al., Neuroscience 136:971-989, 2005; Foda and Marmarou, J Neurosurg 80:301-313, 1994; Kallakuri et al., Exp Brain Res 148:419-424, 2003). Accordingly, use of this preclinical model offers an opportunity for (a) gaining a greater understanding of the relationships of TBI induced diffuse axonal injuries and associated long term disabilities, and (b) to provide a platform for quantitative assessment of treatment interactions upon the trajectories of TBI-induced disabilities. Using the impact acceleration closed head TBI model to induce mild/moderate injuries in the rat, we have observed and quantitated multiple morbidities commonly observed following TBI in humans (Bose et al., J Neurotrauma 30:1177-1191, 2013). This chapter describes methods and protocols used for TBI-induced multiple morbidity involving cognitive dysfunction, balance instability, spasticity and gait, and anxiety-like disorder.

Prolonged hippocampal cell death following closed-head traumatic brain injury in rats.

Traumatic brain injury (TBI) leads to enduring cognitive disorders. Although recent evidence has shown that controlled cortical impact in a rodent may induce memory deficits with prolonged cell death in the dentate gyrus (DG) of the hippocampus, few studies have reported long-term chronic hippocampal cell death following 'closed-head' TBI (cTBI), the predominant form of human TBI. Therefore, the aim of this study was to quantify terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)(+) apoptotic hippocampal cells as well as hippocampal cells with hallmark morphological features of degenerating cells in a chronic setting of cTBI in rats. TUNEL assays and Cresyl violet staining were performed using 6-month post-TBI fixed hippocampal sections. Evidence of prolonged hippocampal cell death was shown by the presence of a significantly increased number of TUNEL(+) cells in the cornu ammonis 1-3 (CA1-CA3) and DG of the hippocampus compared with intact controls. In addition, Cresyl violet staining indicated a significantly elevated number of cells with the degenerative morphological features in all hippocampal subregions (CA1-CA3, hilus, and DG). These results suggest that prolonged cell death may occur in multiple regions of the hippocampus following cTBI.

Trigeminal neuroplasticity underlies allodynia in a preclinical model of mild closed head traumatic brain injury (cTBI).

Post-traumatic headache (PTH) following TBI is a common and often persisting pain disability. PTH is often associated with a multimodal central pain sensitization on the skin surface described as allodynia. However, the particular neurobiology underlying cTBI-induced pain disorders are not known. These studies were performed to assess trigeminal sensory sensitization and to determine if sensitization measured behaviorally correlated with detectable changes in portions of the trigeminal sensory system (TSS), particularly trigeminal nucleus, thalamus, and sensory cortex. Thermal stimulation is particularly well suited to evaluate sensitization and was used in these studies. Recent advances in the use of reward/conflict paradigms permit use of operant measures of behavior, versus reflex-driven response behaviors, for thermal sensitization studies. Thus, to quantitate facial thermal sensitization (allodynia) in the setting of acute TBI, the current study utilized an operant orofacial pain reward/conflict testing paradigm to assess facial thermal sensitivity in uninjured control animals compared with those two weeks after cTBI in a rodent model. Significant reductions in facial contact/lick behaviors were observed in the TBI animals using either cool or warm challenge temperatures compared with behaviors in the normal animals. These facial thermal sensitizations correlated with detectable changes in multiple levels of the TSS. The immunohistochemical (IHC) studies revealed significant alterations in the expression of the serotonin (5-HT), neurokinin 1 receptor (NK1R), norepinephrine (NE), and gamma-aminobutyric acid (GABA) in the caudal trigeminal nucleus, thalamic VPL/VPM nucleus, and sensory cortex of the orofacial pain pathways. There was a strong correlation between increased expression of certain IHC markers and increased behavioral markers for facial sensitization. The authors conclude that TBI-induced changes observed in the TSS are consistent with the expression of generalized facial allodynia following cTBI. To our knowledge, this is the first report of orofacial sensitization correlated with changes in selected neuromodulators/neurotransmitters in the TSS following experimental mild TBI.

Effect of combined treadmill training and magnetic stimulation on spasticity and gait impairments after cervical spinal cord injury.

Spasticity and gait impairments are two common disabilities after cervical spinal cord injury (C-SCI). In this study, we tested the therapeutic effects of early treadmill locomotor training (Tm) initiated at postoperative (PO) day 8 and continued for 6 weeks with injury site transcranial magnetic stimulation (TMSsc) on spasticity and gait impairments after low C6/7 moderate contusion C-SCI in a rat model. The combined treatment group (Tm+TMSsc) showed the most robust decreases in velocity-dependent ankle torques and triceps surae electromyography burst amplitudes that were time locked to the initial phase of lengthening, as well as the most improvement in limb coordination quantitated using three-dimensional kinematics and CatWalk gait analyses, compared to the control or single-treatment groups. These significant treatment-associated decreases in measures of spasticity and gait impairment were also accompanied by marked treatment-associated up-regulation of dopamine beta-hydroxylase, glutamic acid decarboxylase 67, gamma-aminobutyric acid B receptor, and brain-derived neurotrophic factor in the lumbar spinal cord (SC) segments of the treatment groups, compared to tissues from the C-SCI nontreated animals. We propose that the treatment-induced up-regulation of these systems enhanced the adaptive plasticity in the SC, in part through enhanced expression of pre- and postsynaptic reflex regulatory processes. Further, we propose that locomotor exercise in the setting of C-SCI may decrease aspects of the spontaneous maladaptive segmental and descending plasticity. Accordingly, TMSsc treatment is characterized as an adjuvant stimulation that may further enhance this capacity. These data are the first to suggest that a combination of Tm and TMSsc across the injury site can be an effective treatment modality for C-SCI-induced spasticity and gait impairments and provided a pre-clinical demonstration for feasibility and efficacy of early TMSsc intervention after C-SCI.

Effects of acute intrathecal baclofen in an animal model of TBI-induced spasticity, cognitive, and balance disabilities.

Spasticity is a major health problem for patients with traumatic brain injury (TBI). In addition to spasticity, TBI patients exhibit enduring cognitive, balance, and other motor impairments. Although the use of antispastic medications, particularly ITB, can decrease the severity of TBI-induced spasticity, current guidelines preclude the use of ITB during the first year after TBI. Therefore, the present study was performed to quantitate disability in an animal model of closed-head TBI (cTBI; Mararou's model) after ITB treatment. After cTBI, significant deficits in spasticity and gait, cognitive, balance, and anxiety-like behaviors were detected. ITB (Lioresal(®)) or saline was administered using Alzet pumps (0.8 µg/hour for 4 weeks). Spasticity measures using velocity-dependent ankle torque and ankle extensor muscle electromyography recordings, footprints (gait), balance performance tests, serial learning, and anxiety-like behaviors were performed at multiple post-treatment and -withdrawal of ITB time points. Our data indicated that 1 month of ITB treatment initiated at post-TBI week 1 blocked the early onset of spasticity and significantly attenuated late-onset spasticity and anxiety-like behavior with no significant adverse effects on cognitive and balance performance. This improved spasticity outcome was accompanied by marked up-regulation of gamma-aminobutyric acid (GABA)/GABAb, norepinephrine, and brain-derived neurotrophic factor expression in spinal cord tissue. Early intervention with ITB treatment was safe, feasible, and effective in this cTBI animal model. Collectively, these data provide a strong molecular footprint of enhanced expression of reflex regulation by presynaptic inhibition. The possibility that acute ITB treatment may decrease maladaptive segmental and descending plasticity is discussed. The data provided by the present animal model initiates a pre-clinical platform for safety, feasibility, and efficacy of early ITB intervention after TBI.

Altered patterns of reflex excitability, balance, and locomotion following spinal cord injury and locomotor training.

Spasticity is an important problem that complicates daily living in many individuals with spinal cord injury (SCI). While previous studies in human and animals revealed significant improvements in locomotor ability with treadmill locomotor training, it is not known to what extent locomotor training influences spasticity. In addition, it would be of considerable practical interest to know how the more ergonomically feasible cycle training compares with treadmill training as therapy to manage SCI-induced spasticity and to improve locomotor function. Thus the main objective of our present studies was to evaluate the influence of different types of locomotor training on measures of limb spasticity, gait, and reflex components that contribute to locomotion. For these studies, 30 animals received midthoracic SCI using the standard Multicenter Animal Spinal cord Injury Studies (MASCIS) protocol (10 g 2.5 cm weight drop). They were divided randomly into three equal groups: control (contused untrained), contused treadmill trained, and contused cycle trained. Treadmill and cycle training were started on post-injury day 8. Velocity-dependent ankle torque was tested across a wide range of velocities (612-49°/s) to permit quantitation of tonic (low velocity) and dynamic (high velocity) contributions to lower limb spasticity. By post-injury weeks 4 and 6, the untrained group revealed significant velocity-dependent ankle extensor spasticity, compared to pre-surgical control values. At these post-injury time points, spasticity was not observed in either of the two training groups. Instead, a significantly milder form of velocity-dependent spasticity was detected at postcontusion weeks 8-12 in both treadmill and bicycle training groups at the four fastest ankle rotation velocities (350-612°/s). Locomotor training using treadmill or bicycle also produced significant increase in the rate of recovery of limb placement measures (limb axis, base of support, and open field locomotor ability) and reflex rate-depression, a quantitative assessment of neurophysiological processes that regulate segmental reflex excitability, compared with those of untrained injured controls. Light microscopic qualitative studies of spared tissue revealed better preservation of myelin, axons, and collagen morphology in both locomotor trained animals. Both locomotor trained groups revealed decreased lesion volume (rostro-caudal extension) and more spared tissue at the lesion site. These improvements were accompanied by marked upregulation of BDNF, GABA/GABA(b), and monoamines (e.g., norepinephrine and serotonin) which might account for these improved functions. These data are the first to indicate that the therapeutic efficacy of ergonomically practical cycle training is equal to that of the more labor-intensive treadmill training in reducing spasticity and improving locomotion following SCI in an animal model.

Delayed synaptic transmission in Drosophila cacophonynull embryos.

Ca(2+) influx through the Drosophila N-type Ca(2+) channel, encoded by cacophony (cac), triggers fast synaptic transmission. We now ask whether the cac Ca(2+) channel is the Ca(2+) channel solely dedicated for fast synaptic transmission. Because the cac(null) mutation is lethal, we used cac(null) embryos to address this question. At the neuromuscular junction in HL3 solution, no fast synchronous synaptic transmission was detected on nerve stimulation. When the wild-type cac gene was introduced in the cac(null) background, fast synaptic transmission recovered. However, even in cac(null) embryos, nerve stimulation infrequently induced delayed synaptic events in the minority of cells in 1.5 mM [Ca(2+)](e) and in the majority of cells in 5 mM [Ca(2+)](e). The number of delayed quantal events per stimulus was greater in 5 mM [Ca(2+)](e) than in 1.5 mM. Thus the delayed release is [Ca(2+)](e) dependent. Plectreurys toxin II (PLTXII) (10 nM; a spider toxin analog) depressed the frequency of delayed events, suggesting that voltage-gated Ca(2+) channels, other than cac Ca(2+) channels, are contributing to them. However, delayed events were not affected by 50 microM La(3+). The frequency of miniature synaptic currents in cac(null) embryos was approximately 1/2 of control, whereas in high K(+) solutions, it was approximately 1/135. The hypertonicity response was approximately 1/10 of control. These findings indicate that the number of release-ready vesicles is smaller in cac(null) embryos. Taken together, the cac Ca(2+) channel is indispensable for fast synaptic transmission in normal conditions, and another type of Ca(2+) channel, the non-cac, PLTXII-sensitive Ca(2+) channel, is contributing to delayed release in cac(null) embryos.

Nerve-evoked synchronous release and high K+ -induced quantal events are regulated separately by synaptotagmin I at Drosophila neuromuscular junctions.

The distal Ca(2+)-binding domain of synaptotagmin I (Syt I), C2B, has two Ca(2+)-binding sites. To study their function in Drosophila, pairs of aspartates were mutated to asparagines and the mutated syt I was expressed in the syt I-null background (P[syt I(B-D1,2N)] and P[syt I(B-D3,4N)]). We examined the effects of these mutations on nerve-evoked synchronous synaptic transmission and high K(+)-induced quantal events at embryonic neuromuscular junctions. The P[syt I(B-D1,2N)] mutation virtually abolished synaptic transmission, whereas the P[syt I(B-D3,4N)] mutation strongly reduced but did not abolish it. The quantal content in P[syt I(B-D3,4N)] increased with the external Ca(2+) concentration, [Ca(2+)](e), with a slope of 1.86 in double-logarithmic plot, whereas that of control was 2.88. In high K(+) solutions the quantal event frequency in P[syt I(B-D3,4N)] increased progressively with [Ca(2+)](e) between 0 and 0.15 mM as in control. In contrast, in P[syt I(B-D1,2N)] the event frequency did not increase progressively between 0 and 0.15 mM and was significantly lower at 0.15 than at 0.05 mM [Ca(2+)](e). The P[syt I(B-D1,2N)] mutation inhibits high K(+)-induced quantal release in a narrow range of [Ca(2+)](e) (negative regulatory function). When Sr(2+) substituted for Ca(2+), nerve-evoked synchronous synaptic transmission was severely depressed and delayed asynchronous release was appreciably increased in control embryos. In high K(+) solutions with Sr(2+), the quantal event frequency was higher than that in Ca(2+) and increased progressively with [Sr(2+)](e) in control and in both mutants. Sr(2+) partially substitutes for Ca(2+) in synchronous release but does not support the negative regulatory function of Syt I.

Repetitive exposures to nicotine induce a hyper-responsiveness via the cAMP/PKA/CREB signal pathway in Drosophila.

Nicotine, in addition to acute effects, has long-lasting effects on mammalian behaviors, such as those leading to addiction. Here we present genetic and pharmacological evidence in Drosophila suggesting that repetitive exposures to nicotine induce a hyper-responsiveness through synthesis of new protein(s) via CREB-mediated gene transcription. Single exposure to volatilized nicotine dose-dependently inhibited the startle-induced climbing response. Compared with this effect of nicotine in wild-type flies, it was stronger in dunce, which has defective phosphodiesterase, and in wild-type flies treated with a phosphodiesterase inhibitor, whereas it was weaker in DC0, which has defective protein kinase A (PKA), and in wild-type flies treated with a PKA blocker. Thus, the effect of nicotine is enhanced by a mechanism involving the cAMP/PKA cascade. However, in wild-type flies, an increase in head cAMP was not detected within 2 min after single exposure to nicotine, during which the nicotine effect on the behavior was maximal. In wild-type flies, after repetitive exposures to nicotine, the nicotine effect was significantly enhanced and the head cAMP was elevated. The responsiveness to nicotine at second exposure increased with a 4 h interval but not with a 2 h interval, suggesting that the observed hyper-responsiveness was not due to accumulation of residual nicotine. Both enhancement of the nicotine effect and elevation of cAMP during repetitive exposures to nicotine were blocked by a protein synthesis inhibitor. Induction of a dominant negative CREB transgene also blocked the enhancement, suggesting that CREB-mediated gene transcription is required for the hyper-responsiveness.