Computed tomography of ununited anconeal process in the dog.

OBJECTIVE: The purpose of this study was to describe computed tomography (CT) features of the ununited anconeal process and relate them with the following elbow dysplasia signs: medial coronoid disease, medial humeral condyle changes, osteoarthritis (OA), and radioulnar incongruence. METHODS: Computed tomographic images of dogs older than six months with an ununited anconeal process were evaluated (n = 13). Ununited anconeal process features were described as being complete or incomplete, and the degree of displacement, volume, and presence of cysts and sclerosis were also evaluated. Medial coronoid disease was defined as an irregular medial coronoid process shape, presence of sclerosis and fragmentation. Medial humeral condyle changes were defined as subchondral bone flattening, lucencies, and sclerosis. Osteoarthritis was graded depending on the osteophytes size. Radioulnar incongruence was measured on a sagittal view at the base of the medial coronoid process. RESULTS: Eleven elbows had a complete and two had an incomplete ununited anconeal process. All ununited anconeal processes had cystic and sclerotic lesions. Seven ununited anconeal processes were displaced and six were non-displaced. Mean ununited anconeal process volume was 1.35 cm3 (0.61 cm³ - 2.08 cm³). Twelve elbows had signs of medial coronoid disease (4 of them with a fragmented medial coronoid process), and one elbow did not show any evidence of medial coronoid disease. Ten elbows had medial humeral condyle changes. One elbow had grade 1 OA, seven elbows had grade 2, and five elbows grade 3. All elbows had radioulnar incongruence: three elbows had a negative and 10 elbows had a positive radioulnar incongruence. Mean radioulnar incongruence was 1.49 mm (0.63 mm - 2.61 mm). Computed tomographic findings were similar in the majority of the elbows studied: complete ununited anconeal processes with signs of medial coronoid disease, positive radioulnar incongruence, high grade of OA, sclerotic medial humeral condyle changes, and large ununited anconeal process volumes. CLINICAL SIGNIFICANCE: Incomplete small ununited anconeal process volumes could be associated with a lower incidence of medial coronoid disease or medial humeral condyle changes. We recommend performing preoperative CT of elbows with an ununited anconeal process to evaluate concurrent lesions.

Contrast-enhanced ultrasonography in the differentiation of retinal detachment and vitreous membrane in dogs and cats.

OBJECTIVES: To evaluate contrast-enhanced ultrasonography (CEU) and colour Doppler imaging (CDI) for detection of persistent vascularisation in retinal detachment. METHODS: In 22 eyes, retinal detachment (n=13) and vitreous membranes (n=9) were confirmed by ophthalmological examination, during cataract surgery, by histopathology or after vitreoretinal surgery. Tentative diagnosis of retinal detachment or vitreous membrane was made using grey-scale B-mode ultrasonography. Assessment of retinal detachment was based on the presence or absence of vascularisation in the membranous structure using CDI and CEU. RESULTS: Sensitivity, specificity, positive-predictive value and negative-predictive value of grey-scale ultrasonography in differentiating retinal detachment from vitreous membrane were 92·3%, 66·6%, 80% and 85·7%. In 91% of eyes, colour Doppler assessment was unsuccessful due to the movement of the eye. Persistent vascularisation was demonstrated in all cases of retinal detachments with CEU. CEU was 100% accurate for detection and differentiation between retinal detachment and vitreous membrane. CLINICAL SIGNIFICANCE: CEU is a useful clinical tool for the diagnosis of retinal detachment and vitreous membrane in dogs and cats.

Ethanol-induced neuronal apoptosis in vivo requires BAX in the developing mouse brain.

A single episode of ethanol intoxication triggers widespread apoptotic neurodegeneration in the infant rat or mouse brain. The cell death process occurs over a 6-16 h period following ethanol administration, is accompanied by a robust display of caspase-3 enzyme activation, and meets ultrastructural criteria for apoptosis. Two apoptotic pathways (intrinsic and extrinsic) have been described, either of which may culminate in the activation of caspase-3. The intrinsic pathway is regulated by Bax and Bcl-XL and involves Bax-induced mitochondrial dysfunction and release of cytochrome c as antecedent events leading to caspase-3 activation. Activation of caspase-8 is a key event preceding caspase-3 activation in the extrinsic pathway. In the present study, following ethanol administration to infant mice, we found no change in activated caspase-8, which suggests that the extrinsic pathway is not involved in ethanol-induced apoptosis. We also found that ethanol triggers robust caspase-3 activation and apoptotic neurodegeneration in C57BL/6 wildtype mice, but induces neither phenomenon in homozygous Bax-deficient mice. Therefore, it appears that ethanol-induced neuroapoptosis is an intrinsic pathway-mediated phenomenon involving Bax-induced disruption of mitochondrial membranes and cytochrome c release as early events leading to caspase-3 activation.

Apoptosis in the in vivo mammalian forebrain.

Apoptosis is a word originally introduced by Kerr, Wyllie, and colleagues for a cell death process they defined in terms of its ultrastructural appearance in nonneuronal cells from various tissues. There are very few studies providing detailed ultrastructural criteria for recognizing neuronal apoptosis in the in vivo mammalian brain. In the absence of such criteria, the Kerr/Wyllie description pertaining to nonneuronal cells has served as a reference standard. However, contemporary neurobiologists typically rely on cell culture models for studying neuronal apoptosis, and these models are rarely validated ultrastructurally; rather they are assumed to be appropriate models based on unvalidated biochemical tests for apoptosis. Relying on evidence generated in such cell culture models or on nonspecific cytochemical tests applied to brain tissue, many authors have recently suggested that an apoptotic mechanism may mediate neuronal death in a wide variety of human neurodegenerative diseases. Whether the cell death process in neurodegenerative diseases meets ultrastructural criteria for apoptosis has been given very little consideration. Recently, several methods have been described for triggering extensive apoptotic neurodegeneration in the developing in vivo mammalian brain. These methods include head trauma or treatment with several types of drugs (NMDA antagonists, GABAA agonists, or ethanol). We have performed an ultrastructural analysis of the neuronal cell death process triggered in the cerebral cortex and thalamus by these several methods and compared it with physiological cell death (PCD), a prototypic example of neuronal apoptosis that occurs naturally in the developing brain. Our findings, which are reviewed herein, demonstrate that the types and sequence of changes induced by each of the above methods are identical to those that characterize PCD. This confirms that each of these methods produces bona fide in vivo apoptotic neurodegeneration, and it signifies that our description of this neuronal apoptotic process, which differs in some respects from the Kerr/Wyllie description of nonneuronal apoptosis, can serve as a useful reference standard for recognizing the characteristic changes that in vivo neurons undergo when they are dying by an apoptotic mechanism.

Comparison of rat retinal fixation techniques: chemical fixation and microwave irradiation.

In histological studies using retinas, eyes are commonly fixed with aldehyde derivatives administered by immersion or perfusion. However, the histology of rat retinas chemically fixed as a whole eye is typically inferior to the histology of retinas that are immediately fixed after acute dissection from the rest of the eye. Chemical fixation without dissection often results in neuronal swelling resembling excitotoxic damage induced by ischemia because the retina is protected by the sclera and is thus poorly accessible to immersion or perfusion fixation techniques. In order for the acute dissection technique to work properly, it must be completed in a timely manner, which may be difficult under some circumstances. Microwave irradiation is an alternative method for fixing tissues that are inaccessable to chemicals. We examined the effectiveness of microwave irradiation of the whole eye as a substitute for acute retinal dissection. To study the feasibility of microwave methods, we compared retinal morphology using microwave irradiation to morphology using conventional immersion fixation methods. Eyes were removed from rats, placed in a container with 2 or 20 ml artificial cerebrospinal fluid (aCSF) and irradiated with a household microwave oven. For morphological comparison, control eyes were immersed in a chemical fixative containing 1% paraformaldehyde and 1.5% glutaraldehyde. All eyes were embedded in araldite for evaluation by light microscopy. Retinal segments acutely isolated before immersion fixation revealed intact histology whereas retinal segments exposed to 60 min of simulated ischemia showed severe neuronal degeneration. Using an immersion technique, the retinas of chemically fixed whole eyes showed neuronal swelling similar to excitotoxic ischemic damage, suggesting that conventional immersion methods provide poor whole eye fixation. The neuronal degeneration observed with conventional immersion fixation was not found in retinas of whole eyes fixed with 20 sec of microwave irradiation. During microwave irradiation the temperature in the bathing aCSF rose to 55-72 degrees C. In some eyes, overcooking produced chromatin clumping and a small loss of contrast in staining. Although nuclear clumping and diminished staining occasionally result from overcooking, ischemic damage is well controlled with microwave fixation of enucleated eyes. When the optimal conditions are defined, microwave fixation may be preferable for retinal histology if chemical fixation following acute dissection is not feasible.

Swelling of Müller cells induced by AP3 and glutamate transport substrates in rat retina.

Previous studies have shown that a single systemic injection of 2-amino-3-phosphonopropionate (AP3), an agonist/antagonist at metabotropic glutamate receptors, produces marked swelling of rodent Müller cells. To investigate the effects of AP3 on Müller cells, we used in vitro retinal segments prepared from 30 day old rats. Incubation with AP3 for 1 h or more caused severe swelling of Müller cells with the appearance of mitotic cellular profiles in the outer nuclear layer. The Müller cell swelling was mimicked by substrates for glutamate transporters, suggesting that AP3 may produce its effects via transport into glial cells. To determine whether AP3 is a substrate for glutamate transporters, we studied cultured rat hippocampal astrocytes using whole-cell patch clamp recordings. In hippocampal astrocytes, AP3 activated currents via an Na(+)-dependent glutamate transporter. Consistent with this, substitution of extracellular sodium with choline blocked Müller cell swelling in the rat retina. These results indicate that the acute glial swelling produced by AP3 results primarily from a fluid shift that accompanies the transport of AP3 and sodium into Müller cells.

Prevention of trauma-induced neurodegeneration in infant rat brain.

Recent evidence implicates the endogenous excitatory neurotransmitters, glutamate (Glu) and aspartate, in the pathophysiology of traumatic injury in the adult CNS, but it is not known whether similar excitotoxic mechanisms mediate traumatic injury in the immature CNS. Therefore, we developed a model of brain contusion injury in infant rats and used this model to study the nature and evolution of the acute cytopathologic changes and to evaluate the ability of Glu receptor antagonists to protect the immature brain against such changes. Seven-day-old rat pups were subjected to contusion injury and were killed 0, 0.5, 1, 2, 4, and 6 h later for histologic evaluation of the brain. Physical tearing of the dura and minor disruption of underlying brain tissue was noted at 0 h. At 30 min a discrete zone of neuronal necrosis began to appear at the border of the trauma site; this zone progressively expanded over a period of 4 h. The cytopathologic changes closely resembled the type of changes Glu is known to cause; these changes consisted of swollen dendrites, degenerating neurons with pyknotic nuclei and markedly swollen cytoplasm, and dark cells with vacuolated cytoplasm. The noncompetitive N-methyl-D-aspartate (NMDA) antagonist, dizocilpine maleate, when administered 30 min before or 1 h after trauma, significantly attenuated the lesion. The competitive NMDA antagonist, 3-((-2)-carboxypiperazine-4-yl)-propyl-1-phosphonate, was also neuroprotective. The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/kainate receptor antagonist 2,3-dihydro-6-nitro-7-sulfamoyl-benzo(f)quinoxaline did not significantly suppress the lesion when given as three treatments (30 mg/kg each) 30 min before plus 15 and 75 min after the insult. These findings suggest that traumatic injury in the infant rat brain is mediated by endogenous excitotoxins (Glu and aspartate) acting at NMDA receptors and can be substantially mitigated by timely treatment with NMDA receptor antagonists.

Motor neuron degeneration induced by excitotoxin agonists has features in common with those seen in the SOD-1 transgenic mouse model of amyotrophic lateral sclerosis.

A superoxide dismutase 1 (SOD-1)genetic defect has been identified in familial amyotrophic lateral sclerosis (ALS) and motor neuron degeneration has been described in SOD-1 transgenic mice. Because an excitotoxic mechanism has been implicated in ALS, we undertook studies to provide a description of excitotoxic degeneration of spinal motor neurons for comparison with the degenerative process observed in SOD-1 transgenic mice. Excitotoxin agonists selective for each of the three major types of inotropic glutamate receptors were applied directly onto the lumbar spinal cord of 21-day old rats following posterior laminectomy. N-methyl-D-aspartate (NMDA) preferentially affected dorsal horn neurons, whereas the non-NMDA agonist, kainic acid, preferentially affected motor neurons. Cytopathological changes in motor neurons closely resembled those described in SOD-1 mice. These changes consist of massively swollen dendritic processes in the presence of well-preserved presynaptic axon terminals; cell bodies of motor neurons filled with vacuoles that originate both from endoplasmic reticulum and mitochondria; pleomorphic changes in mitochondria; axons of motor neuron becoming swollen proximally with accumulation of vacuoles, organelles, filaments, and degeneration products in the swollen segment. The observed changes in motor axons resemble changes described in the spinal cord of ALS patients. These findings are consistent with the proposal that motor neuron degeneration in ALS may be mediated by an excitotoxic process involving hyperactivation with non-NMDA glutamate receptors.

Age-specific neurotoxicity in the rat associated with NMDA receptor blockade: potential relevance to schizophrenia?

Agents that block the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor induce a schizophrenialike psychosis in adult humans and injure or kill neurons in several corticolimbic regions of the adult rat brain. Susceptibility to the psychotomimetic effects of the NMDA antagonist, ketamine is minimal or absent in children and becomes maximal in early adulthood. We examined the sensitivity of rats at various ages to the neurotoxic effects of the powerful NMDA antagonist, MK-801. Vulnerability was found to be age dependent, having onset at approximately puberty (45 days of age) and becoming maximal in early adulthood. This age-dependency profile (onset of susceptibility in late adolescence) in the rat is similar to that for ketamine-induced psychosis or schizophrenia in humans. These findings suggest that NMDA receptor hypofunction, the mechanism underlying the neurotoxic and psychotomimetic actions of NMDA antagonists, may also play a role in schizophrenia.

An ex vivo rat retinal preparation for excitotoxicity studies.

Although the isolated chicken embryo retina has been a very useful in vitro preparation for studying mechanisms of excitotoxicity, it is an avian rather than mammalian tissue and its embryonic age makes it unsuitable for a full range of developmental and aging studies. Therefore, we have explored the feasibility of using the rat retina at various ages for in vitro excitotoxicity studies. In this model, retinal segments were isolated in artificial cerebrospinal fluid (CSF) at 5 degrees C then incubated under various conditions at 30 degrees C and assessed histologically for signs of neurodegenerative changes. Retinal segments from 7-, 30-, 120- and 660-day-old rats incubated in CSF for 3 h and from 30-day-old rats incubated for 24 h retained a normal histological appearance. Thus, this preparation is suitable for in vitro studies pertaining to either acute or delayed excitotoxic phenomena in the mammalian CNS at any age from infancy to old age. Excitotoxin agonist experiments in the 30-day-old rat retina revealed the surprising result that the non-NMDA agonists, kainate and AMPA, at a low concentration (100 microM) damaged a much larger number of retinal neurons than NMDA did at a very high concentration (10 mM).

Age dependent sensitivity of the rat retina to the excitotoxic action of N-methyl-D-aspartate.

We have found that the rat retina can be isolated atraumatically and incubated ex vivo for up to 24 h without showing signs of histological deterioration, and that retinas from adult or aged rats can be isolated as successfully as those from immature rats. In the present study we used this preparation to show that rat retinal neurones at postnatal day zero (PND 0) are relatively insensitive to the excitotoxic action of the glutamate agonist, N-methyl-D-aspartate (NMDA), then gradually show increasing sensitivity that peaks at about PND 9 and declines from PND 15-30 after which it remains at a low level up to the last time point studied (10 months of age). This is consistent with other developmental NMDA receptor data and underscores the need for caution in using immature in vitro central; nervous system (CNS) tissue preparations as a basis for interpreting the role of NMDA receptors in adult neurological diseases.

Semi-automatic external defibrillation.

Malignant arrhythmia, which is responsible for most of the out-of-hospital cardiac arrests, is ventricular fibrillation (VF). The best treatment of VF is a controlled electric shock on the chest administered in a short delay. The emergency medical technicians (EMTs) qualified to carry out this treatment in Belgium and in districts often succeed in arriving on the spot 8 minutes earlier than the people of the Service Mobile d'Urgence et de Réanimation (SMUR). The delegation of defibrillation to ambulance crew members however implies a specific teaching, training and a medical control. The Brussels experience shows that semi-automatic external defibrillation by EMT-Ds (SAED) is feasible when criteria for applying SAED in the pre-hospital phase are applicable.

Early defibrillation by EMTs: the Brussels experience.

Considering that in Brussels the first-aid ambulance team reaches the patient in cardiac arrest 10 min before the physician-manned ambulance, we instituted a feasibility study of early defibrillation by emergency medical technicians (EMTs). Three hundred EMTs received a 20-h automatic external defibrillation (AED) training course followed by a refresher course every 6 months. Of 316 cardiac arrests included in this study, asystole was encountered in 53% and ventricular fibrillation/ventricular tachycardia (VF/VT) in 33% of the cases on arrival of the EMTs. In the VF/VT group, defibrillation was performed by EMTs with a Laerdal Heartstart 7-9 min before the medical team arrived. The overall cardiac arrest survival rate improved from 7% in 1989 to 19% in 1992. However, the long-term survival rate (14/105) of ventricular fibrillation remained low because of excessive delays in emergency medical service (EMS) access and in early ACLS. In conclusion, this work shows that in Brussels: (1) early defibrillation of cardiac arrest victims in VF is feasible by EMTs when a training and a follow-up program are implemented; (2) the weakest link of the chain of survival is the early EMS access, and the early ACLS; and (3) AED program increases the interest and the efficacy of EMTs and medical teams in the management of cardiac arrests.

NMDA antagonist neurotoxicity: mechanism and prevention.

Antagonists of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor, including phencyclidine (PCP) and ketamine, protect against brain damage in neurological disorders such as stroke. However, these agents have psychotomimetic properties in humans and morphologically damage neurons in the cerebral cortex of rats. It is now shown that the morphological damage can be prevented by certain anticholinergic drugs or by diazepam and barbiturates, which act at the gamma-aminobutyric acid (GABA) receptor-channel complex and are known to suppress the psychotomimetic symptoms caused by ketamine. Thus, it may be possible to prevent the unwanted side effects of NMDA antagonists, thereby enhancing their utility as neuroprotective drugs.

Excitotoxicity of L-dopa and 6-OH-dopa: implications for Parkinson's and Huntington's diseases.

Despite several decades of research aimed at elucidating the mechanisms underlying neuronal degeneration in Parkinson's and Huntington's diseases, these mysteries remain unfathomed. The brain contains high concentrations of the putative transmitters, glutamate and aspartate, which have neurotoxic (excitotoxic) potential and are thought to cause neuronal degeneration in certain acute neurological disorders. However, no mechanism has been identified by which these diffusely distributed agents might cause the regionally selective patterns of neuronal degeneration characterizing Parkinson's and Huntington's diseases. Here we report that L-DOPA, the natural precursor to dopamine, is a weak excitotoxin and its ortho-hydroxylated derivative, 6-OH-DOPA, is a powerful excitotoxin. We propose that an excitotoxic process mediated by L-DOPA or an acidic derivative such as 6-OH-DOPA might be responsible for degeneration of nigral neurons in Parkinson's disease or striatal neurons in Huntington's disease.

L-cysteine, a bicarbonate-sensitive endogenous excitotoxin.

After systemic administration to immature rodents, L-cysteine destroys neurons in the cerebral cortex, hippocampus, thalamus, and striatum, but the underlying mechanism has never been clarified. This neurotoxicity of L-cysteine, in vitro or in vivo, has now been shown to be mediated primarily through the N-methyl-D-aspartate subtype of glutamate receptor (with quisqualate receptor participation at higher concentrations). In addition, the excitotoxic potency of L-cysteine was substantially increased in the presence of physiological concentrations of bicarbonate ion. L-Cysteine is naturally present in the human brain and in the environment, and is much more powerful than beta-N-methylamino-L-alanine, a bicarbonate-dependent excitotoxin, which has been implicated in an adult neurodegenerative disorder endemic to Guam. Thus, the potential involvement of this common sulfur-containing amino acid in neurodegenerative processes affecting the central nervous system warrants consideration.

Sensitivity of the developing rat brain to hypobaric/ischemic damage parallels sensitivity to N-methyl-aspartate neurotoxicity.

The endogenous excitotoxin, glutamate (Glu), acting at the N-methyl-aspartate (NMA) subtype of Glu receptor, is thought to play a major role in hypoxic/ischemic neuronal degeneration. In the present study, the sensitivities of the developing rat CNS to hypoxic/ischemic neuronal degeneration and to the neurotoxic action of NMA were compared at various postnatal ages. In the hypoxic/ischemic experiments, ischemia was produced by unilateral common carotid artery ligation and hypoxia by subjecting the pups to a partial vacuum. Keeping the duration of the hypobaric episode constant at 75 min for all age groups, we observed that the vulnerability of the immature brain to hypobaric/ischemic damage increased during the early neonatal period (days 2-4), reached a peak at day 6 and then diminished progressively with increasing age. In the second part of the study, NMA was microinjected unilaterally into the head of the caudate nucleus at various postnatal ages (2-80 d). In the early neonatal period (days 2-6), injections of relatively small doses of NMA (6-15 nmol) produced a dose-dependent widespread excitotoxic reaction throughout the forebrain with peak sensitivity being observed on day 6. The cytotoxic reaction to NMA was identical in appearance and time course to that induced by hypobaric/ischemic methods. With increasing age, the excitotoxic response to a given dose of NMA decreased progressively and the lesions became more strictly confined to the injection site. Cell populations most sensitive to NMA toxicity in the 2-10 d period closely correlated with those most vulnerable to hypoxia/ischemia, and sensitivity to both types of injury reached a peak at 6 d. These findings reinforce other evidence linking an excitotoxic mechanism and the NMA subtype of Glu receptor to hypoxic/ischemic brain damage and suggest that there may be a period during development when NMA receptors are hypersensitive to excitotoxic stimulation, thus rendering the neurons possessing such receptors hypervulnerable to hypoxic/ischemic damage.

Pathological changes induced in cerebrocortical neurons by phencyclidine and related drugs.

Phencyclidine (PCP), a dissociative anesthetic and widely abused psychotomimetic drug, and MK-801, a potent PCP receptor ligand, have neuroprotective properties stemming from their ability to antagonize the excitotoxic actions of endogenous excitatory amino acids such as glutamate and aspartate. There is growing interest in the potential application of these compounds in the treatment of neurological disorders. However, there is an apparent neurotoxic effect of PCP and related agents (MK-801, tiletamine, and ketamine), which has heretofore been overlooked: these drugs induce acute pathomorphological changes in specific populations of brain neurons when administered subcutaneously to adult rats in relatively low doses. These findings raise new questions regarding the safety of these agents in the clinical management of neurodegenerative diseases and reinforce concerns about the potential risks associated with illicit use of PCP.

Hypobaric-ischemic conditions produce glutamate-like cytopathology in infant rat brain.

We present a new animal model of perinatal hypoxic/ischemic brain damage and compare this type of brain damage with the excitotoxic type of damage previously described in the brains of infant rats and monkeys treated systemically with glutamate (Glu). Ten-d-old rats with unilateral occlusion of the common carotid artery were subjected to hypobaric conditions for 75 min and sacrificed 0-4 hr later for light and electron microscopic brain examination. The mortality rate was relatively low (12%), and brain damage was evident ipsilateral to the ligated carotid in 94% of surviving animals 4 hr after termination of the hypobaric event. Regions most frequently affected were the medial habenulum, dentate gyrus, caudate nucleus, frontoparietal neocortices, olfactory tubercle, and several thalamic nuclei. The acute cytopathological changes, primarily edematous degeneration of neuronal dendrites and cell bodies, evolved very rapidly, with some neurons manifesting end-stage necrosis at 0 hr (immediately after hypobaric exposure) and others developing such changes over a 1-4-hr period. We conclude that the neurodegenerative reaction induced in infant rat brain by hypoxia/ischemia is indistinguishable from the excitotoxic type of damage exogenous Glu is known to cause. Moreover, in a companion study (Olney et al., 1989) we show that MK-801, a powerful antagonist of the N-methyl-D-aspartate receptor complex (subtype of Glu receptor), protects against neuronal degeneration in this hypobaric/ischemic model. Our results reinforce other recent evidence suggesting that hypoxic/ischemic brain damage is mediated by endogenous Glu or related excitotoxins.