Positive Allosteric Modulation of Cholinergic Receptors Improves Spatial Learning after Cortical Contusion Injury in Mice.

We examined benzyl quinolone carboxylic acid (BQCA), a novel M1 muscarinic-positive allosteric modulator, for improving memory and motor dysfunction after cerebral cortical contusion injury (CCI). Adult mice received unilateral motorsensory cortical CCI or sham injury. Benzyl quinolone carboxylic acid (BQCA; 5, 10, and 20 mg/kg, intraperitoneally [i.p.] × 2/day × 3-4 weeks) or vehicle (Veh) were administered, and weekly evaluations were undertaken using a battery of motor tests, as well as the Morris water maze. Thereafter, cerebral metabolic activation was investigated in awake animals during walking with [14C]-2-deoxygIucose autoradiography, comparing CCI mice previously treated with BQCA (20 mg/kg) or vehicle. Relative changes in local cerebral glucose uptake (rCGU) were evaluated in three-dimensional-reconstructed brains using statistical parametric mapping. CCI resulted in mild hyperactivity in the open field, and modest significant motor deficits, as well as significantly decreased spatial learning at 3 weeks. BQCA in CCI mice resulted in significantly improved spatial recall during the third week, with minimal effects on motor outcomes. CCI significantly decreased rCGU in the ipsilesional basal ganglia-thalamocortical circuit and in somatosensory regions, with relative increases noted contralaterally, as well as in the cerebellum. Significant decreases in rCGU were noted in subregions of the ipsilesional hippocampal formation, with significant increases noted contralesionally. BQCA compared to vehicle-treated mice showed modest, though significantly increased, rCGU in motor regions, as well as a partial reversal of lesion-related rCGU findings in subregions of the hippocampal formation. rCGU in ipsilesional posterior CA1 demonstrated a significant inverse correlation with latency to find the submerged platform. BQCA at 20 mg/kg had no significant effect on general motor activity, body weight, or acute motor, secretory, or respiratory symptoms. Results suggest that BQCA is a candidate compound to improve learning and memory function after brain trauma and may not suffer the associated central nervous system side effects typically associated with even modest doses of other cholinergic enhancers.

Thyroid hormone treatment activates protective pathways in both in vivo and in vitro models of neuronal injury.

Thyroid hormone plays an important role in brain development and adult brain function, and may influence neuronal recovery after Traumatic Brain Injury (TBI). We utilized both animal and cell culture models to determine the effects of thyroid hormone treatment, post TBI or during hypoxia, on genes important for neuronal survival and neurogenesis. We show that TBI in rats is associated with a reduction in serum thyroxine (T4) and triiodothyronine (T3). A single dose of levothyroxine (T4), one hour after injury, increased serum T4 and normalized serum T3 levels. Expression of genes important for thyroid hormone action in the brain, MCT8 and Type 2 deiodinase (Dio2) mRNA, diminished after injury, but were partially restored with T4 treatment. mRNA from the Type 3 deiodinase (Dio3) gene, which inactivates T4 to reverse T3 (rT3), was induced 2.7 fold by TBI, and further stimulated 6.7-fold by T4 treatment. T4 treatment significantly increased the expression of mRNA from Bcl2, VEGFA, Sox2 and neurotrophin, genes important for neuronal survival and recovery. The cortex, compared to the hippocampus and cerebellum, sustained the greatest injury and had the most significant change in gene expression as a result of injury and the greatest response to T4 treatment. We utilized hypoxia to study the effect of neuronal injury in vitro. Neuroblastoma cells were exposed to reduced oxygen tension, 0.2%, and were compared to cells grown at control oxygen levels of 21%. T3 treatment significantly increased hypoxia inducible factor (HIF)-2α protein, but not HIF-1α. In a hypoxia time course exposure, expression of hypoxia-mediated genes (VEGF, Enolase, HIF2α, c-Jun) peaked at least 8 h earlier with T3-treatment, compared to cells grown without T3. The early induction of these genes may promote cellular growth after injury. After hypoxic injury, T3 induced mRNA expression of the genes, KLF9 and hairless, important for T3-mediated brain function. The findings from both in vitro and in vivo studies support a role of thyroid hormone in activating pathways important for neuronal protection and promotion of neuronal recovery after injury.

Dry-contact microelectrode membranes for wireless detection of electrical phenotypes in neonatal mouse hearts.

Continuous monitoring of aberrant electrical rhythms during heart injury and repair requires prolonged data acquisition. We hereby developed a wearable microelectrode membrane that could be adherent to the chest of neonatal mice for in situ wireless recording of electrocardiogram (ECG) signals. The novel dry-contact membrane with a meshed parylene-C pad adjacent to the microelectrodes and the expandable meandrous strips allowed for varying size of the neonates. The performance was evaluated at the system level; specifically, the ECG signals (µV) acquired from the microelectrodes underwent two-stage amplification, band-pass filtering, and optical data transmission by an infrared Light Emitting Diode (LED) to the data-receiving unit. The circuitry was prototyped on a printed circuit board (PCB), consuming less than 300 µW, and was completely powered by an inductive coupling link. Distinct P waves, QRS complexes, and T waves of ECG signals were demonstrated from the non-pharmacologically sedated neonates at ~600 beats per minutes. Thus, we demonstrate the feasibility of both real-time and wireless monitoring cardiac rhythms in a neonatal mouse (17-20 mm and <1 g) via dry-contact microelectrode membrane; thus, providing a basis for diagnosing aberrant electrical conduction in animal models of cardiac injury and repair.

Neurologic dysfunction and genotoxicity induced by low levels of chlorpyrifos.

Chlorpyrifos (CPF) is an organophosphorus cholinesterase inhibitor widely used as an insecticide. Neuro and genotoxicity of this agent were evaluated following daily subcutaneous injections at 0.1, 1 and 10mg/kg or its vehicle to laboratory rats during one week, at the end of which somatosensory evoked potentials (SEP) and power spectrum of the electroencephalogram (EEGp) were recorded under urethane anesthesia. In another group of conscious animals, auditory startle reflex (ASR) was evaluated followed, after euthanasia, with measurements of plasma B-esterases, and genotoxicity with the alkaline comet assay (ACA) at the same CPF doses. The results indicated a CPF dose related inhibition of B-esterases. Enhanced inhibition of the ASR by a subthreshold pre-pulse was observed at all doses and ACA showed a significant higher DNA damage than vehicle controls in animals exposed to 10mg/kg CPF. A trend to higher frequencies of EEGp and an increase in amplitude of the first negative wave of the SEP were found at all doses. The first positive wave of the SEP decreased at the CPF dose of 10mg/kg. In summary, a shift to higher EEG frequencies and alterations of somatosensory and auditory input to the central nervous system were sensitive manifestations of CPF toxicity, associated with depression of B-esterases. The changes in electrical activity of the cerebral cortex and DNA damage observed at doses that do not elicit overt toxicity may be useful in the detection of CPF exposure before clinical signs appear.

Arginine NO-dependent and NO-independent effects on hemodynamics.

L-arginine administration decreases mean arterial blood pressure (MABP), presumably by excess nitric oxide (NO) synthesis. However, some reports indicate that d-arginine, not a substrate of NO synthase (NOS), also induces hypotension. To clarify this phenomenon, the hemodynamic effects of L- and D-arginine and their modification by NOS inhibition with L-nitroarginine methyl ester (L-NAME) were assessed. MABP, cardiac output, stroke volume, heart rate and systemic vascular resistance were recorded in Sprague-Dawley rats under urethane or ketamine/diazepam anesthesia, with or without blockade of NO synthesis by L-NAME. Both stereoisomers of arginine induced a dose-related drop in MABP of similar magnitude and time course, but recovery from hypotension was slower in L-arginine than in D-arginine. The hypotension induced by both stereoisomers was due to a decrease in systemic vascular resistance (SVR) with increase in cardiac output (CO) and stroke volume (SV). Administration of L-NAME induced a pronounced increase in MABP and SVR, with decreases in CO and heart rate (HR). Infusion of L-arginine after L-NAME significantly decreased MABP and SVR at the highest dose while d-arginine failed to do so. After L-NAME, MABP was significantly lower under l-arginine than under d-arginine at all doses. These experiments suggest a dual mechanism in the hypotensive effect of L-arginine: a NO independent action on vascular resistance shared with D-arginine, and a NO dependent mechanism that becomes evident in the presence of NOS inhibition with L-NAME. Cardiac effects of NO do not appear to play a role in L-arginine hypotension.

Exercise training reinstates cortico-cortical sensorimotor functional connectivity following striatal lesioning: development and application of a subregional-level analytic toolbox for perfusion autoradiographs of the rat brain.

Current rodent connectome projects are revealing brain structural connectivity with unprecedented resolution and completeness. How subregional structural connectivity relates to subregional functional interactions is an emerging research topic. We describe a method for standardized, mesoscopic-level data sampling from autoradiographic coronal sections of the rat brain, and for correlation-based analysis and intuitive display of cortico-cortical functional connectivity (FC) on a flattened cortical map. A graphic user interface "Cx-2D" allows for the display of significant correlations of individual regions-of-interest, as well as graph theoretical metrics across the cortex. Cx-2D was tested on an autoradiographic data set of cerebral blood flow (CBF) of rats that had undergone bilateral striatal lesions, followed by 4 weeks of aerobic exercise training or no exercise. Effects of lesioning and exercise on cortico-cortical FC were examined during a locomotor challenge in this rat model of Parkinsonism. Subregional FC analysis revealed a rich functional reorganization of the brain in response to lesioning and exercise that was not apparent in a standard analysis focused on CBF of isolated brain regions. Lesioned rats showed diminished degree centrality of lateral primary motor cortex, as well as neighboring somatosensory cortex-changes that were substantially reversed in lesioned rats following exercise training. Seed analysis revealed that exercise increased positive correlations in motor and somatosensory cortex, with little effect in non-sensorimotor regions such as visual, auditory, and piriform cortex. The current analysis revealed that exercise partially reinstated sensorimotor FC lost following dopaminergic deafferentation. Cx-2D allows for standardized data sampling from images of brain slices, as well as analysis and display of cortico-cortical FC in the rat cerebral cortex with potential applications in a variety of autoradiographic and histologic studies.

Repeated blast exposure alters open field behavior recorded under low illumination.

Blast-induced traumatic brain injury (bTBI) can have devastating behavioral consequences. This study was designed to evaluate the behavioral consequences of single or repeated bTBI, as evaluated by an open field (OF) test conducted in near-darkness to avoid confounding effects of illumination and photophobia. Sprague-Dawley rats under isoflurane anesthesia were exposed to a series of 3 sub-lethal blasts into a compressed air-driven blast chamber separated by 2 week intervals (n=11). Sham controls received anesthesia but without blast exposure (n=11). OF tests were performed 1 or 7 days after each blast using a computerized video tracking system in near-darkness to monitor spontaneous activity. Spatial and temporal variables calculated for both blast and sham groups were: Distance moved (cm) and time (s) spent in the center or periphery zones of the field, total distance traveled, speed in center and periphery zones, rearing events and non-linear regressions of distance moved and rearing events on time. Results showed that the sham group expressed the expected decrease (habituation) in total distance walked, and distance walked as well as speed in center and periphery in successive exposures to the OF while the blast group did not, a sign of impaired learning. The blast group also walked more and faster and demonstrated more rearing behavior, both considered OF signs of anxiety. These results indicate that OF outcomes of bTBI in animals have resemblance to alterations observed in human subjects with this condition and might be useful in evaluating the response of behavioral outcomes of bTBI to experimental treatments.

Remote brain network changes after unilateral cortical impact injury and their modulation by acetylcholinesterase inhibition.

We explored whether cerebral cortical impact injury (CCI) effects extend beyond direct lesion sites to affect remote brain networks, and whether acetylcholinesterase (AChE) inhibition elicits discrete changes in functional activation of motor circuits following CCI. Adult male rats underwent unilateral motor-sensory CCI or sham injury. Physostigmine (AChE inhibitor) or saline were administered subcutaneously continuously via implanted minipumps (1.6 micromoles/kg/day) for 3 weeks, followed by cerebral perfusion mapping during treadmill walking using [(14)C]-iodoantipyrine. Quantitative autoradiographs were analyzed by statistical parametric mapping and functional connectivity (FC) analysis. CCI resulted in functional deficits in the ipsilesional basal ganglia, with increased activation contralesionally. Recruitment was also observed, especially contralesionally, of the red nucleus, superior colliculus, pedunculopontine tegmental nucleus, thalamus (ventrolateral n., central medial n.), cerebellum, and sensory cortex. FC decreased significantly within ipsi- and contralesional motor circuits and between hemispheres, but increased between midline cerebellum and select regions of the basal ganglia within each hemisphere. Physostigmine significantly increased functional brain activation in the cerebellar thalamocortical pathway (midline cerebellum→ventrolateral thalamus→motor cortex), subthalamic nucleus/zona incerta, and red nucleus and bilateral sensory cortex. In conclusion, CCI resulted in increased functional recruitment of contralesional motor cortex and bilateral subcortical motor regions, as well as recruitment of the cerebellar-thalamocortical circuit and contralesional sensory cortex. This phenomenon, augmented by physostigmine, may partially compensate motor deficits. FC decreased inter-hemispherically and in negative, but not positive, intra-hemispherical FC, and it was not affected by physostigmine. Circuit-based approaches into functional brain reorganization may inform future behavioral or molecular strategies to augment targeted neurorehabilitation.

Acetylcholinesterase inhibition interacts with training to reverse spatial learning deficits after cortical impact injury.

Cholinergic mechanisms are known to play a key role in cognitive functions that are profoundly altered in traumatic brain injury (TBI). The present investigation was designed to test the ability of continuous administration, starting at the time of injury, of physostigmine (PHY), an acetylcholinesterase (AChE) inhibitor that crosses the blood-brain barrier (BBB), to ameliorate the alterations of learning and memory induced by cerebral cortex impact injury in rats under isoflurane anesthesia. Learning and memory were assessed with the Morris water maze implemented during days 7-11 (WM1), and days 21-25 post-TBI (WM2), with four trials per day for 3 days, followed by target reversal and 2 additional days of training. These groups of Sprague-Dawley male rats were used: TBI treated with PHY at 3.2 µmol/kg/day (TBI-PHY3.2), or 6.4 µmol/kg/day (TBI-PHY6.4), by subcutaneous osmotic pumps, or TBI and no injury (Sham) treated with saline. AChE activity was measured in brain tissue samples of non-traumatized animals that received PHY at the doses used in the TBI animals. In WM1 tests, PHY3.2 improved learning within sessions, but not between sessions, in the recall of the target position, while PHY6.4 had no significant effects. In WM2 tests, PHY improved within- and between-sessions performance at both dose levels. We found that continuous AChE inhibition interacted with repeated training on the water maze task to completely reverse the deficits seen in learning and memory induced by TBI. The PHY treatment also reduced the amount of brain tissue loss as measured using cresyl violet staining.

Calf tissue oxygenation during exercise in men with and without risk factors for developing peripheral arterial disease.

OBJECTIVE: This study aimed to compare calf tissue oxygenation responses to calf exercise in men without diagnosed peripheral arterial disease but with selected risk factors for peripheral arterial disease with those without risk factors. DESIGN: A cross-sectional quasi-experimental design was used. The no-risk group (n = 20) had none of the risk factors (diabetes, hypertension, hyperlipidemia, obesity, current or 10 pack-yr smoking history, or age ≥65 yrs). The at-risk group (n = 45) had one to six risk factors. Medial calf tissue oxygenation (percentage saturation) was determined using near-infrared spectroscopy during seven consecutive 5-min test stages: rest, 0-W active plantar/dorsiflexion, rest, 4-W resistive plantar flexion, rest, 8-W resistive plantar flexion, and rest. Resistive exercise was performed on the Stresst'er calf ergometer. RESULTS: Compared with the no-risk group, decrements in calf tissue oxygenation induced by light-to-moderate resistive calf exercise in the at-risk group was significantly greater (by 9% saturation) (4-W: P < 0.001; 8-W: P = 0.002). CONCLUSIONS: Men with risk factors for developing peripheral arterial disease but without such diagnosis demonstrated greater decrements in calf tissue oxygenation during calf exercise compared with men without risk factors. Further development of this test may lead to early diagnosis and intervention to modify risk factors and prevent co-morbidities.

Acetylcholinesterase inhibition and locomotor function after motor-sensory cortex impact injury.

Traumatic brain injury (TBI) induces transient or persistent dysfunction of gait and balance. Enhancement of cholinergic transmission has been reported to accelerate recovery of cognitive function after TBI, but the effects of this intervention on locomotor activity remain largely unexplored. The hypothesis that enhancement of cholinergic function by inhibition of acetylcholinesterase (AChE) improves locomotion following TBI was tested in Sprague-Dawley male rats after a unilateral controlled cortical impact (CCI) injury of the motor-sensory cortex. Locomotion was tested by time to fall on the constant speed and accelerating Rotarod, placement errors and time to cross while walking through a horizontal ladder, activity monitoring in the home cages, and rearing behavior. Assessments were performed the 1st and 2nd day and the 1st, 2nd, and 3rd week after TBI. The AChE inhibitor physostigmine hemisulfate (PHY) was administered continuously via osmotic minipumps implanted subcutaneously at the rates of 1.6-12.8 µmol/kg/day. All measures of locomotion were impaired by TBI and recovered to initial levels between 1 and 3 weeks post-TBI, with the exception of the maximum speed achievable on the accelerating Rotarod, as well as rearing in the open field. PHY improved performance in the accelerating Rotarod at 1.6 and 3.2 µmol/kg/day (AChE activity 95 and 78% of control, respectively), however, higher doses induced progressive deterioration. No effect or worsening of outcomes was observed at all PHY doses for home cage activity, rearing, and horizontal ladder walking. Potential benefits of cholinesterase inhibition on locomotor function have to be weighed against the evidence of the narrow range of useful doses.

The environmental pollutant endosulfan disrupts cerebral cortical function at low doses.

Endosulfan can induce convulsions that could lead to brain damage. The variability and lack of specificity of neurological signs and symptoms in the pre-convulsive stages makes early diagnosis difficult. We sought to determine if electrophysiological exploration of the cerebral cortex could yield objective signs of endosulfan intoxication at levels that do not elicit convulsions. Endosulfan was administered intravenously to Sprague-Dawley adult rats under urethane anesthesia at doses from 0.5 to 4mg/kg. EEG power and the evoked potentials (EP) to forepaw electrical stimulation were studied over the contralateral (S1CL) and homolateral (S1HL) cortical somatosensory areas and the contralateral visual area (V1CL). At each area, five EP waves were measured. Arterial blood pressure, heart rate and body temperature were also recorded. Endosulfan induced a dose-related increase in EPs at all sites. At S1CL, EP peak amplitude was greater than baseline at 1, 2 and 4mg/kg for the first negative, second positive and third negative waves, and at 2 and 4mg/kg for the first and third positive waves. Similar but less marked trends were observed at S1HL and V1CL. A shift of EEG power to higher frequencies (alpha and beta EEG bands) was only present at 4mg/kg. In conclusion, endosulfan induced a large increase of cortical evoked potentials amplitudes at doses that did not elicit convulsions. These responses could be used as a non-invasive diagnostic tool to detect low-level endosulfan intoxication in humans and to help establish the NOAEL and LOAEL levels of this pollutant.

Ergometric performance during exercise training in men with intermittent claudication.

OBJECTIVE: To determine and describe changes in weekly work, power, exercise times, and recovery times during an exercise training intervention in men with peripheral arterial disease (PAD) and intermittent calf claudication. DESIGN: Tracking of weekly exercise training parameters involved repeated measures over time in one group of participants. Other outcomes of this pilot study used a one-group, pretest-posttest design. SETTING: Tertiary-care medical center. PARTICIPANTS: Fifteen male veterans (mean age, 69 years) with Fontaine stage IIa PAD and classic intermittent calf claudication. MAIN OUTCOME MEASUREMENTS: Participants completed graded treadmill exercise tests before and after intervention from which maximal walking power was calculated. Work, power, and exercise and recovery times for each exercise training session were computed and averaged for each week. INTERVENTION: The intervention consisted of an intensive 3-month exercise training program involving walking and calf muscle exercises: 3 sessions per week at the clinic (treadmill walking and calf ergometry) and 2 sessions per week at home (free walking and standing heel raises). RESULTS: After training, participants increased treadmill maximal walking power from 220 to 414 W (by 87%). Treadmill and calf exercise work, power, and exercise time per session increased linearly during 13 weeks of training, whereas recovery time per session of treadmill exercise decreased. During the same period, treadmill and calf exercise training power outputs increased by averages of 227% and 92%, respectively. CONCLUSION: Calculation of work and power during exercise training can be used to track progress quantitatively at short intervals. Weekly linear increases in training work and power per exercise session suggest that optimal intervention duration may be longer than 3 months for men with PAD and intermittent calf claudication.

Preamputation evaluation of lower-limb skeletal muscle perfusion with H(2) (15)O positron emission tomography.

OBJECTIVE: To establish whether muscle blood flow (MBF) measurements with O-water positron emission tomography could reliably identify patients with critical limb ischemia and detect and quantify a distal deficit in skeletal MBF in these cases. DESIGN: O-water positron emission tomography scans were performed at rest or during unloaded ankle plantar and dorsiflexion exercise of the diseased leg in 17 subjects with leg ischemia or on a randomly selected leg of 18 age-matched healthy control subjects. TcPO2 was evaluated with Novametrix monitors and perfusion of skin topically heated to 44 degrees C and adjacent nonheated areas with a Moor Instruments laser Doppler imaging scanner. RESULTS: The enhancement of MBF induced by exercise was significantly lower in ischemic than in normal legs, and the sensitivity and specificity of this phenomenon were similar to those of laser Doppler imaging or TcPO2 in identifying ischemia subjects. In addition, the exercise MBF deficit was predominant at the distal-leg levels, indicating the ability of the technique to help determine the correct level of amputation. CONCLUSIONS: Skeletal MBF of legs with severe ischemia can be detected accurately with O-water positron emission tomography and could add valuable information about viability of skeletal muscle in the residual limb when deciding the level of an amputation.

Scanning laser-Doppler imaging of leg- and foot-skin perfusion in normal subjects: analysis of age, gender, site, and laser-type effects.

OBJECTIVES: To report normal values of skin perfusion in healthy subjects in three age groups using a laser Doppler imager; to determine differences attributable to gender, age, site, and use of red or near-infrared lasers; and to correlate transcutaneous oxygen with laser flux values. DESIGN: Flux and transcutaneous oxygen were measured at ten sites in the lower extremity in 60 subjects from three age groups. Heated and unheated sites were scanned with red and near-infrared lasers. RESULTS: Heat hyperemia was prominent at all sites. Small, statistically significant mean +/- SD differences were found between heated and nonheated sites for the red and near-infrared lasers (P = 0.02). All flux ratios were independent of gender but were higher in the oldest group. Plantar sites demonstrated higher flux in unheated areas and lower flux ratios compared with leg sites. Transcutaneous oxygen did not correlate significantly with flux for either laser type. CONCLUSIONS: Scanning laser-Doppler imaging flux values provide a reference for identifying patients at risk for tissue ischemia and poor healing potential caused by impaired circulatory reserve in the legs and distal feet. The lack of correlation between flux and transcutaneous oxygen in healthy individuals suggests that they measure different physiologic processes.

Pilot studies to demonstrate that intestinal mucosal afferent nerves are functionally linked to visceral adipose tissue.

Dietary capsaicin reduces rodent visceral fat weight. We tested the hypothesis that intact intestinal mucosal afferent nerve function is necessary for fat deposition in visceral adipose tissue sites. Rats were treated daily for 2 weeks with intragastric (chronic treatment) vehicle or capsaicin. Superior mesenteric artery blood flow and mesenteric and inguinal fat blood flow were measured before and after capsaicin was administered into the duodenum (acute treatment). Fat from all sites was dissected and weighed. Chronic capsaicin significantly attenuated acute capsaicin-induced mesenteric hyperemia but did not abolish the reflex wiping of the eye exposed to capsaicin, indicating that functional ablation was limited to the intestinal mucosal afferent nerves. The associated vasoconstriction in adipose tissue was inhibited at the visceral (mesenteric) site and maintained but attenuated at the subcutaneous (inguinal) site. The onset of vasoconstriction was instantaneous, indicating a reflex mechanism. There was a redistribution of fat from visceral to subcutaneous sites, reflected by a decrease and an increase in the percentage of body fat in the visceral and subcutaneous sites, respectively. These pilot studies reveal for the first time that normal intestinal mucosal afferent nerve function is necessary for the physiologic accumulation of fat in visceral adipose tissue sites.

Acetylcholine and choline dynamics provide early and late markers of traumatic brain injury.

We assessed acetylcholine (ACh) and choline (Ch) dynamics 2.5 h, 1, 4 and 14 days after cerebral cortex impact injury or craniotomy only in adult male Sprague-Dawley rats. Cortical endogenous ACh (D0ACh), endogenous free Ch (D0Ch), deuterium-labeled Ch (D4Ch), and ACh synthesized from D4Ch (D4ACh) were measured by gas-chromatography mass-spectrometry after intravenous injection of D4Ch followed in 1 min by microwave fixation of the brain. D0Ch increased in and around the impact up to 700% of control within 1 day after trauma. Smaller D0Ch increases were found in the cortex contralateral to the impact and in both hemispheres after craniotomy only. D4Ch contents increased to 200% in the impact and surrounding regions 4-14 days post-trauma, with lower increases 2.5 h post-trauma. D0ACh decreased at all times post-trauma in the impact center, and initially in the periphery and adjacent regions with a recovery at 14 days. Similar D0ACh decreases, although of lesser extent and magnitude were present in the craniotomy only group. D4ACh showed a peak at one day post-trauma in all regions studied in the impact and craniotomy groups. In conclusion, D0Ch tissue level was an early marker of trauma, while 14 days after trauma Ch uptake from blood was enhanced in and around the traumatized cortex. Craniotomy by itself induced a generalized increase in ACh turnover 1 day after this minimal trauma. Choline acetyltransferase activity was reduced in the impact center region but not affected in the adjacent and contralateral regions or by craniotomy.

Circadian rhythms of heart rate and locomotion after treatment with low-dose acetylcholinesterase inhibitors.

This study tested the hypothesis that repeated exposure to low levels of sarin, pyridostigmine bromide (PB) or their combination, at doses equivalent to those possibly experienced by veterans of the 1991 Persian Gulf War, could lead to persistent or delayed autonomic effects and thus help to explain the cause of clinical findings in this population. Male Sprague-Dawley rats were treated for 3 weeks with: saline injection (0.5 ml kg(-1), s.c., 3 times weekly) with tap drinking water (control); saline injection with PB (80 mg l(-1) in drinking water); sarin injection (62.5 microg kg(-1), s.c., 0.5 x LD(50), 3 times weekly) with tap drinking water (sarin); or sarin injection with PB in drinking water (sarin + PB). At 2, 4 or 16 weeks post-treatment, heart rate (HR) and locomotor activity (LA) were studied by radiotelemetry. Two weeks posttreatment, HR in drug-treated animals was significantly lower than in controls. A decrease in low-frequency HR power spectrum (PS) was found at 00:00 h and 08:00 h with sarin + PB and at 00:00 h with sarin, while total power was enhanced with sarin + PB at 22:00 h. Minimal effects of drug treatments on HR and HR PS were detected at 4 and 16 weeks post-treatment. No significant differences in LA between control and other groups were found. Since no consistent long-term effects were found in any of the variables studied, these experiments do not support the hypothesis that repeated administration of low doses of PB and the nerve agent sarin can induce persistent or delayed alterations in autonomic function.

Cerebral acetylcholine and choline contents and turnover following low-dose acetylcholinesterase inhibitors treatment in rats.

Male Sprague-Dawley rats were treated for 3 weeks with (1) regular tap drinking water plus subcutaneous (s.c.) saline (0.5 ml/kg) injections three times/week, (2) pyridostigmine bromide (PB) in drinking water (80 mg/L) plus s.c. saline injections three times/week, (3) regular tap drinking water plus s.c. sarin (0.5 x LD(50)) injections three times/week, or (4) PB in drinking water plus s.c. sarin injections three times/week. Repeated doses of sarin, in the presence or absence of PB, were devoid of acute toxicity during the three-week treatment period. Two, 4, and 16 weeks post-treatment, animals were given an intravenous pulse injection of choline labeled with 4 deuterium atoms (D4Ch) followed, after 1 min, by microwave fixation of the brain in vivo. Tissue levels of endogenous acetylcholine (D0ACh), endogenous choline (D0Ch), D4Ch, and ACh synthesized from D4Ch (D4ACh) were measured by gas-chromatography mass-spectrometry in hippocampus, infundibulum, mesencephalon, neocortex, piriform cortex, and striatum. Ch uptake from blood and ACh turnover were estimated from D4Ch and D4ACh concentrations in brain tissue, respectively. Statistically significant differences among brain regions were found for D0Ch, D4Ch, D0ACh and D4ACh at 2, 4 and 16 weeks post-treatment. However, differences in the values of these parameters between control and drug treatments were found only for D0ACh and D0Ch at 2 and 4 weeks, but not at 16 weeks post-treatment. In conclusion, the results from these experiments do not support a delayed or persistent alteration in cholinergic function after exposure to low doses of PB and/or sarin.