Theophylline treatment improves mitochondrial function after upper cervical spinal cord hemisection.

The importance of mitochondria in spinal cord injury has mainly been attributed to their participation in apoptosis at the site of injury. But another aspect of mitochondrial function is the generation of more than 90% of cellular energy in the form of ATP, mediated by the oxidative phosphorylation (OxPhos) process. Cytochrome c oxidase (CcO) is a central OxPhos component and changes in its activity reflect changes in energy demand. A recent study suggests that respiratory muscle function in chronic obstructive pulmonary disease (COPD) patients is compromised via alterations in mitochondrial function. In an animal model of cervical spinal cord hemisection (C2HS) respiratory dysfunction, we have shown that theophylline improves respiratory function. In the present study, we tested the hypothesis that theophylline improves respiratory function at the cellular level via improved mitochondrial function in the C2HS model. We demonstrate that CcO activity was significantly (33%) increased in the spinal cord adjacent to the site of injury (C3-C5), and that administration of theophylline (20mg/kg 3x daily orally) after C2HS leads to an even more pronounced increase in CcO activity of 62% compared to sham-operated animals. These results are paralleled by a significant increase in cellular ATP levels (51% in the hemidiaphragm ipsilateral to the hemisection). We conclude that C2HS increases energy demand and activates mitochondrial respiration, and that theophylline treatment improves energy levels through activation of the mitochondrial OxPhos process to provide energy for tissue repair and functional recovery after paralysis in the C2HS model.

Amelioration of hypoperfusion after traumatic brain injury by in vivo endothelin-1 knockout.

Endothelin 1 (ET-1) is one of the most powerful vasoconstrictors in the brain. Its expression is upregulated after traumatic brain injury (TBI) and is a major factor in the ensuing hypoperfusion. Attenuation of ET-1 effects has been mainly achieved by blockade of its receptors. The result of a direct blockade of ET-1 mRNA synthesis is not known. We used the Marmarou's model to inflict injury to male Sprague-Dawley rats injected with antisense ET-1 oligodeoxynucleotides (ODNs) before injury. Laser Doppler flowmetry in noninjured rats (2 groups, i.e., untreated and animals that received cODNs) revealed a constant cerebral blood flow of approximately 14 mL.min-1.100 g-1, whereas the values from injured animals pretreated with control ODNs (cODNs) or from animals subjected to TBI alone were approximately 8.0 mL.min-1.100 g-1 during the 18-48 h time period post-TBI. After antisense ET-1 ODNs pretreatment, however, cerebral blood flow in injured animals was approximately 17 mL.min-1.100 g-1 during the 6-48 h time period. Antisense ET-1 ODNs-treated animals also had 19%-29% larger microvessel cross-sectional area and approximately one-third less ET-1 immunoreactivity in the 50-75% range after injury than did cODNs-treated animals after TBI. The results indicate that this direct in vivo approach is an effective therapeutic intervention for the restoration of cerebral blood flow after TBI.

Differential expression of adenosine A1 and A2A receptors after upper cervical (C2) spinal cord hemisection in adult rats.

BACKGROUND: In an animal model of spinal cord injury, a latent respiratory motor pathway can be pharmacologically activated via adenosine receptors to restore respiratory function after cervical (C2) spinal cord hemisection that paralyzes the hemidiaphragm ipsilateral to injury. Although spinal phrenic motoneurons immunopositive for adenosine receptors have been demonstrated (C3-C5), it is unclear if adenosine receptor protein levels are altered after C2 hemisection and theophylline administration. OBJECTIVE: To assess the effects of C2 spinal cord hemisection and theophylline administration on the expression of adenosine receptor proteins. METHODS: Adenosine A1 and A2A receptor protein levels were assessed in adult rats classified as (a) noninjured and theophylline treated, (b) C2 hemisected, (c) C2 hemisected and administered theophylline orally (3x daily) for 3 days only, and (d) C2 hemisected and administered theophylline (3x daily for 3 days) and assessed 12 days after drug administration. Assessment of A1 protein levels was carried out via immunohistochemistry and A2A protein levels by densitometry. RESULTS: Adenosine A1 protein levels decreased significantly (both ipsilateral and contralateral to injury) after C2 hemisection; however, the decrease was attenuated in hemisected and theophylline-treated animals. Attenuation in adenosine A1 receptor protein levels persisted when theophylline administration was stopped for 12 days prior to assessment. Adenosine A2A protein levels were unchanged by C2 hemisection; however, theophylline reduced the levels within the phrenic motoneurons. Furthermore, the decrease in A2A levels persisted 12 days after theophylline was withdrawn. CONCLUSION: Our findings suggest that theophylline mitigates the effects of C2 hemisection by attenuating the C2 hemisection-induced decrease in A1 protein levels. Furthermore, A2A protein levels are unaltered by C2 hemisection but decrease after continuous or interrupted theophylline administration. The effects on protein levels may underlie the stimulant actions of theophylline.

Alterations in cerebral cortex microvessels and the microcirculation in a rat model of traumatic brain injury: a correlative EM and laser Doppler flowmetry study.

OBJECTIVES: We sought to establish the temporal association of fluctuations in cortical cerebral blood flow (CBF) with ultrastructural alterations of microvessels in rat sensorimotor cortex (smCx) following administration of a rodent acceleration impact model of traumatic brain injury (TBI). METHODS: Laser Doppler flowmetry (LDF) and electron microscopy (EM) were used in parallel experiments that lasted for up to 48 hours after induction of TBI. RESULTS: Compared to sham-operated control, there was a 37% reduction of cortical CBF between 12 and 24 hours, this reduction remaining unchanged for up to 48 hours post-TBI. Ultrastructural alterations in the lumen and wall of smCx microvessels, including endothelial cell distortion and luminal collapse, were seen at hour 1 and continued up to 48 hours after trauma. Compared to control, there was a 40% decrease in the average microvascular luminal area 4 hours and a trend to recover (21%) by 48 hours after trauma. Smooth muscle (SM) in the wall of reacting microvessels showed evidence of increase contractility that coincided temporally with the decreased perfusion of cortical CBF. DISCUSSION: Based on these observations, it is proposed that TBI causes alterations in the vascular tone of reacting microvessels which leads to prolonged vasoreactivity and restriction of the lumen in many but not all microvessels.

Spatial alterations in endothelin receptor expression are temporally associated with the altered microcirculation after brain trauma.

OBJECTIVES: To study the cellular distribution of endothelin receptors A and B (ETrA and ETrB) in the post-traumatic sensorimotor cortex and hippocampus. MATERIALS AND METHODS: We inflicted closed head trauma to male Sprague-Dawley rats and visualized ETrA and ETrB immunoreactivity with 3,3'-diaminobenzidine. RESULTS: ETrA immunolabeling was the most prominent in pyramidal neurons 24 and 48 hours post-trauma, while it reached its peak in the microvasculature at hour 4. ETrB immunolabeling was observed in endothelial cells, perivascular neurons, smooth muscle cells (SM) and pericytes, the expression being the most pronounced 24 hours post-trauma. DISCUSSION: The results suggest that the vasoconstrictor effect of endothelin-1 (ET-1) is mediated primarily by ETrA. The dual effects of ETrB are reflected in its vasoconstrictor role at the vascular bed and conversely, in the attenuation of ET-1 availability and synthesis. We conclude that both receptors play a role in the disturbed microvascular autoregulation and in the sustained reduction of blood flow following trauma to the brain.

Subcellular redistribution of calponin underlies sustained vascular contractility following traumatic brain injury.

OBJECTIVES: The purpose of this study was to observe temporal changes in calponin (Cp), a contractile protein, in response to traumatic brain injury (TBI). METHODS: Double immunocytochemistry in conjunction with morphometric methods was used to study Cp temporal migration in smooth muscle cells (SM) of reacting microvessels following TBI, as induced using a weight-drop, acceleration impact method. RESULTS: Quantification of migrated Cp in the SM wall after TBI was carried out on three-dimensional orthographic reconstructions of serial, digitally acquired images and optical densitometry. Color shifts in Cp intensity were measured in three arbitrary longitudinal compartments, luminal (lu), middle (m) and abluminal (ablu), of SM cytoplasm with respect to proximity to the vessel's lumen. By 24 and 48 hours after TBI, most Cp had migrated from the SM compartment closest to the lu to that farthest away or ablu. In addition, a qualitative increase in Cp was detected closest to the ablu compartment in those segments of the vessel severely constricted. DISCUSSION: Cp migration from cytoskeletal to contractile regions of SM supports its role both in the initiation of vessel contractility and its interaction with cytoskeletal structures subjacent to the cell membrane in SM's contracted state.

In vivo measurement of tissue damage, oxygen saturation changes and blood flow changes after experimental traumatic brain injury in rats using susceptibility weighted imaging.

Traumatic brain injury (TBI) is a prevalent disease, and many TBI patients experience disturbed cerebral blood flow (CBF) after injury. Moreover, TBI is difficult to quantify with conventional imaging modalities. In this paper, we utilized susceptibility weighted imaging (SWI) as a means to monitor functional blood oxygenation changes and to quantify CBF changes in animals after trauma. In this study using six rats, brain trauma was induced by a weight drop model and the brain was scanned over four time points: pre trauma, and 4 h, 24 h and 48 h post trauma. Five rats survived and one died after trauma. A blood phase analysis using filtered SWI phase images suggested that three rats recovered after 48 h and two rats deteriorated. SWI also suggested that CBF decreased by up to 26%. The CBF change is in agreement with the results of arterial spin labeling methods conducted in this study and with previously published results. Furthermore, SWI revealed an enlargement of the major venous vasculature in deep brain structures, in accordance with the location of diffuse axonal injury. Compared with the traditional, invasive, clinical monitoring of cerebral vascular damage and reduction in blood flow, this method offers a novel, safe and noninvasive approach to quantify changes in oxygen saturation and CBF and to visualize structural changes in blood vasculature after TBI.

Calponin and caldesmon cellular domains in reacting microvessels following traumatic brain injury.

Calponin (Cp) and caldesmon (Cd) are actin-binding proteins involved in the regulation of smooth muscle (SM) tone during blood vessel contraction. While in vitro studies have reported modifications of these proteins during vessel contractility, their role in vivo remains unclear. Traumatic brain injury (TBI) causes disruption of cerebral microvascular tone, leading to sustained contractility in reacting microvessels and cerebral hypoperfusion. This study aimed to determine the spatial and temporal expressions of Cp and Cd in rat cerebral cortical and hippocampal microvessels post-TBI. Reacting microvessels were analyzed in control, 4, 24, and 48 h post-injury. Single and double immunocytochemical techniques together with semiquantitative analyses revealed a Cp upregulation in SM at all time frames post-TBI; with the protein migrating from SM cytosol to the vicinity of the cell membrane. Similarly, Cd immunoreactivity significantly increased in both SM and endothelial cells (En). However, while Cp and Cd in SM remained elevated, their levels in En returned to normal at 48 h post-TBI. The results suggest that Cp and Cd levels increase while compartmentalizing to specific subcellular domains. These changes are temporally associated with modifications in the cytoskeleton and contractile apparatus of SM and En during blood vessel contractility. Furthermore, these changes may underlie the state of sustained contractility and hypoperfusion observed in reacting microvessels after TBI.

Cellular compartmentalization of phosphorylated eIF2alpha and neuronal NOS in human temporal lobe epilepsy with hippocampal sclerosis.

Hippocampal sclerosis (HS) is the most common neuropathologic finding in patients with medically refractory temporal lobe epilepsy (TLE). The mechanisms resulting in neuronal injury and cell loss in HS are incompletely understood, but inhibition of protein synthesis may play a pivotal role in these processes. This study examined the relationships between two molecules known to be involved in reduced protein synthesis in animals subjected to traumatic brain injury. Translational initiation of protein synthesis is inhibited when 2alpha (eIF2alpha) is phosphorylated. Recently, nitric oxide (NO) has been shown to reduce protein synthesis by inducing phosphorylation of eIF2alpha. We performed immunocytochemistry for eIF2alpha(P) and histochemistry (NADPH-D reaction) for nitric oxide synthase (NOS) to determine the distribution of these molecules in hippocampi removed from patients undergoing anterior temporal lobectomy (ATL) for medically intractable TLE due to HS. The greatest number of eIF2alpha(P) positive cells was in the CA1 sector of the hippocampus, followed by the hilus of the dentate gyrus. NADPH-D positive neurons were observed most often in the hilus. Labeling in both instances involved neuronal cell body cytoplasm and varicose processes. Combination of both staining procedures revealed close relationships between differentially labeled neurons within the hilus. The results suggest that NO participates in the phosphorylation of eIF2alpha since we demonstrated that nNOS processes are closely related to eIF2alpha(P) positive cells. This may occur through activation of kinases such as PERK, which was recently revealed. In human, TLE protein synthesis inhibition may occur at the translational level since the eIF2alpha (P) labeling is cytoplasmic. Protein synthesis inhibition may contribute to neuronal cell injury and death in HS.