Dynamic respiratory endoscopy of Standardbred racehorses during qualifying races.

REASON FOR PERFORMING STUDY: Examination of the equine upper airway during racing has not previously been documented. OBJECTIVE: To describe the feasibility and appearance of the upper airways by overground respiratory endoscopic examination during racing conditions. METHODS: Overground videoendoscopic examinations were performed on 46 Standardbred racehorses during qualifying races. Examined horses' speeds were recorded throughout the race with a portable GPS device. RESULTS: The procedure did not interfere with performance as there were no significant differences in race times between races in which horses were examined with the endoscope in place and prior unexamined races. Airway obstructions during or after the race were documented in 21 horses. Most previously reported causes of upper airway obstruction were observed; surprisingly bilateral ventro-medial arytenoid displacement (VMAD; n = 5) was seen during exercise as frequently as dorsal displacement of the soft palate (DDSP). Although DDSP (n = 10) was the most common diagnosis made, many displacements (n = 5) occurred after the race. Horses that demonstrated DDSP after the race had slower speeds than unaffected horses during the race. CONCLUSIONS: Racing endoscopy permits the diagnosis of upper airway obstructions without affecting performance. The occurrence of DDSP immediately after exercise may be clinically relevant. During racing VMAD may be an important anomaly. POTENTIAL RELEVANCE: Racing endoscopy could be used to correlate the sensitivity of diagnostic endoscopy during race-training or treadmill examination. The pathogenesis and significance of VMAD deserves further investigation.

Targeting protein kinase C: new therapeutic opportunities against high-grade malignant gliomas?

A large body of evidence suggests that the abnormal phenotype of neoplastic astrocytes, including their excessive proliferation rate and high propensity to invade surrounding tissues, results from mutations in critical genes involved in key cellular events. These genetic alterations can affect cell-surface-associated receptors, elements of signaling pathways, or components of the cell cycle clock, conferring a gain or a loss of relevant metabolic functions of the cells. The understanding of such phenomena may allow the development of more efficacious forms of cancer treatment. Examples are therapies specifically directed against overexpressed epidermal growth factor receptor, hyperactive Ras, excessively stimulated Raf-1, overproduced ornithine decarboxylase, or aberrantly activated cyclin-dependent kinases. The applicability of some of these approaches is now being assessed in patients suffering from primary malignant central nervous system tumors that are not amenable to current therapeutic modalities. Another potentially useful therapeutic strategy against such tumors involves the inhibition of hyperactive or overexpressed protein kinase C (PKC). This strategy is justified by the decrease in cell proliferation and invasion following inhibition of the activity of this enzyme observed in preclinical glioma models. Thus, interference with PKC activity may represent a novel form of experimental cancer treatment that may simultaneously restrain the hyperproliferative state and the invasive capacity of high-grade malignant gliomas without inducing the expected toxicity of classical cytotoxic agents. Of note, the experimental use of PKC-inhibiting agents in patients with refractory high-grade malignant gliomas has indeed led to some clinical responses. The present paper reviews the current status of the biochemistry and molecular biology of PKC, as well as the possibilities for developing novel anti-PKC-based therapies for central nervous system malignancies.

5-Aminolevulinic acid inhibits [3H]muscimol binding to human and rat brain synaptic membranes.

The interaction of 5-aminolevulinic acid (ALA) with GABA(A) receptors has been proposed to underlie the neurological dysfunctions of ALA-accumulating disorders, such as acute intermittent porphyria. The effects of ALA on [3H]muscimol binding to human and rat cerebral cortical membranes were compared. ALA (0.1-10 mM) significantly inhibited the binding of [3H]muscimol (12 nM), with a similar potency in rat and human membranes (IC50 = 199 vs. 228 microM, respectively). Kinetical analysis revealed that ALA (1 mM) significantly increased the Kd and decreased the Bmax of [3H]muscimol to both rat (100 and 50%, respectively) and human (200 and 40%, respectively) membranes, indicating a mixed-type inhibition. The similarity in the potency and mechanism of the ALA-induced inhibition of muscimol binding in rat and human membranes indicate that rat studies are useful to evaluate the neurotoxic properties of ALA towards the human GABAergic system, and may help to understand the pathophysiology of porphyria.

Neurochemical characterization of traumatic brain injury in humans.

Trauma is the leading cause of death in individuals between the ages of 1 and 44 years. And, in the case of severe head injury mortality can reach as high as 35-70%. Despite this fact, there has been little progress in the development of effective pharmacological agents to protect brain injured patients. To date, there is little data on the mechanisms involved in neuronal cellular insult after severe head injury, especially in humans. Glutamate acts both as a primary excitatory neurotransmitter and a potential neurotoxin within the mammalian brain. Evidence indicates that hyperactivity of the glutamate system contributes to neuronal death in brain trauma. Also, in animal models of neurotrauma, this neural injury is followed by gliosis which has been linked to the severity of brain injury. To investigate the glutamate system in brain trauma, we carried out [3H]glutamate and [3H]MK801 (a noncompetitive NMDA-receptor antagonist) binding and [3H]glutamate uptake assays in human cerebral cortex preparations obtained from severely brain injured and control victims. Additionally, to investigate gliosis following brain injury, we performed GFAP immunohistochemistry. There were no significant differences in [3H]glutamate binding (affinity or density of sites) between the control and head injured groups. In contrast, cerebral cortical [3H]MK801 binding revealed both a significant increase in the density of sites (Bmax) and a decrease in the dissociation constant (Kd) in the head injured group when compared to controls. There were no significant differences in [3H]glutamate uptake between groups. The injured brains presented an increased number of GFAP-positive astrocytes and more intense GFAP reaction in comparison to control brains. In the context of traumatic brain injury, our results encourage further investigation into compounds capable of selective modulation of NMDA receptor subtype in humans while also therapeutically manipulating glial cell responses following brain trauma.

Increased serum S100beta protein concentrations following severe head injury in humans: a biochemical marker of brain death?

This study investigated S100beta protein as a biochemical serum marker of brain damage in severe head injury and brain death victims. Blood samples obtained from 15 patients with severe head injury admitted to the trauma intensive care unit (ICU), five patients with a diagnosis of brain death due to hemorrhage following cerebral aneurysm rupture, and five healthy individuals were investigated. The S100beta protein serum concentrations were analyzed with a immunoradiometric assay kit. The 15 patients with severe head injury were followed up for 6 months. Outcome was considered either death or recovery with ICU discharge. S100beta concentrations were closely related to brain damage. Among the severe head injury victims, higher S100beta concentrations were detected in those patients that progressed to death. The individuals with brain death had similar mean S100beta concentrations, irrespective of its cause (either trauma or vascular rupture). S100beta protein is a promising serum outcome predictor for severe head injury victims and may contribute to the early diagnosis of brain death.

Inhibition of adenylate cyclase activity by 5-aminolevulinic acid in rat and human brain.

The effect of the haem precursor 5-aminolevulinic acid (ALA) on the production of cyclic adenosine-monophosphate (cAMP) by rat cerebellar membranes was investigated. It was found that ALA dose-dependently decreased cAMP levels (maximal inhibition of 38%, at 1 mM), due to an inhibition of basal adenylate cyclase activity. ALA also inhibited fluoride- and Gpp(NH)p-stimulated, but not the forskolin-stimulated adenylate cyclase activity. 5-Aminovaleric acid (an inhibitor of GABA(B) receptors) did not prevent the inhibition, indicating that it was not mediated by the activation of the G(i)-protein coupled GABA(B) receptor. In addition, the nucleotide binding site of G-protein appeared not to be affected by ALA since it did not inhibit [3H]Gpp(NH)p binding to our membrane preparation. Antioxidants (glutathione, ascorbate and trolox) completely prevented the inhibition indicating that ALA effect was mediated by an oxidative damage of adenylate cyclase. ALA also inhibited the activity of adenylate cyclase in membranes isolated from rat cortex and striatum and from human cortex. These results may be of value in understanding the neurochemical mechanisms underlying the neurotoxic effects of ALA.

Effects of lead on adenylate cyclase activity in rat cerebral cortex.

Lead decreased in a dose dependent manner the basal AC activity in membranes of rat cerebral cortex (IC50 = 2.5 +/- 0.1 microM). In membranes preincubated under basal conditions, AC activity was stimulated by approximately two and fourfold by 10 microM Gpp(NH)p or forskolin, respectively. Under basal conditions, lead (3 microM) inhibited enzyme activity up to 50%, but was not able to inhibit the Gpp(NH)p- or the forskolin-stimulated AC activity. However, in membranes preincubated with Gpp(NH)p (10 microM), lead (3 microM) had no significant effect on enzyme activity, but it partly blocked the stimulation of AC activity elicited by forskolin (10 microM). In membranes preincubated with 10 microM lead, the addition of 10 microM Gpp(NH)p or forskolin in the incubation medium did not stimulate AC activity. However, when added together in the incubation medium Gpp(NH)p + forskolin produced an increase in enzyme activity. In membranes preincubated with 10 microM lead + 10 microM Gpp(NH)p, Gpp(NH)p (10 microM) or forskolin (10 microM) added alone or in combination to the incubation medium did not stimulate AC activity. Moreover, under these latter conditions lead had no further effect on enzyme activity. These results indicate that lead may interact with G-proteins and with the catalytic subunit of cerebral cortical AC to produce inhibition of the enzyme activity.

Effects of guanine nucleotides on glutamate-induced chemiluminescence in rat hippocampal slices submitted to hypoxia.

Glutamate significantly increased levels of spontaneous chemiluminescence (CL) in rat hippocampal slices incubated under hypoxic conditions. Although it has been previously shown that guanine nucleotides (GN) displace glutamate from several of its receptors, in our study only GMP, as well as the glutamate antagonist MK-801, was able to reverse the increase in CL provoked by glutamate. On the other hand, not only GTP or Gpp(NH)p failed to reverse the action of glutamate, but they increased CL production like glutamate. This effect of GTP/Gpp(NH)p was also reversed by GMP. We concluded that, under neurotoxic conditions, GMP acted as an antagonist and GTP or Gpp(NH)p acted as agonists of glutamate. These results reinforced the evidence of the existence of extracellular site(s) for GN and indicated a possible role for GN in excitotoxicity.

Guanine nucleotides are present in human CSF.

Several studies have shown that experimentally added guanine nucleotides (GN) may extracellularly modulate the glutamatergic system. However, there is no previous report of the extracellular occurrence of GN in the CNS. This study used HPLC to investigate the presence of GN in cerebrospinal fluid (CSF) samples of 26 patients. We demonstrated the extracellular presence of GN in the CNS. In human CSF, GMP was detected in a remarkably high concentration (236.20 microM). This evidence stimulates further investigation of extracellular GN modulation of neurotransmission, physiological mechanisms of action(s), and therapeutic potential(s) in the CNS.