Air-filled ultrasound contrast agents do not damage the cerebral microvasculature or brain tissue in rats.

RATIONALE AND OBJECTIVES: Air microemboli may damage the cerebral microvasculature. The aim of this study was to evaluate the safety of ultrasound contrast agents composed of air microspheres with regard to cerebral damage when administered into the arterial system (ie, when not filtered by the capillary system of the lungs). METHODS: Three experimental methods were used in 75 rats after injection of either Albunex, Echovist, or Levovist into the left heart ventricle. The alkaline phosphatase (ALP) method to demonstrate small segmental brain capillary and arteriolar dilatations (SCADs), intravenous injections of Evans blue and fluorescence microscopy to detect increased vascular permeability (blood-brain barrier damage), and histologic examination of the brain to detect microinfarction. Intracardiac injections of saline, air, and corn oil were used as controls. RESULTS: Brain microinfarcts and SCADs formation of the brain microvasculature occurred only after control injections with corn oil. None of the brains from animals that received ultrasound contrast agent showed gross discoloration, as an indication of increased vascular permeability, with the Evans blue/fluorescence microscopy method. Definite leakage of Evans blue occurred only after large doses (150 microL) of air. CONCLUSIONS: This study indicates that ultrasound contrast media composed of air microspheres do not cause lesions of the brain microvasculature or parenchyma.

Macrocephaly, epilepsy, autism, dysmorphic features, and mental retardation in two sisters: a new autosomal recessive syndrome?

We report two sisters with macrocephaly, epilepsy, and severe mental retardation. The first child was a 14 year old girl born at term after a normal pregnancy, with birth weight 3600 g and occipitofrontal circumference (OFC) 36 cm (75th centile). Her head size increased markedly during the first six months of life, and was later stable at 2-3 cm above the 97.5th centile. Her development was characterised by psychomotor delay, epilepsy, and autistic features. Her face appeared mildly dysmorphic with a large forehead, short philtrum, and bushy eyebrows. Her younger sister was also born at term with birth weight 2600 g and OFC 34 cm (25th centile). She also developed postnatal macrocephaly with OFC 2 cm above the 97.5th centile and the same mild dysmorphic facial features as her sister. Her development was also characterised by psychomotor delay, autistic features, and epilepsy. In addition, she suffered from coeliac disease. She died unexpectedly at the age of 5 years, probably from an epileptic attack. Necropsy confirmed megalencephaly but no other pathological changes were found. The clinical features in these two sisters do not fit with any known syndrome and may represent a previously unrecognised autosomal recessive disorder.

Apolipoprotein E genotype and Alzheimer's disease in an elderly Norwegian cohort.

Apolipoprotein E (Apo E) genotyping was performed on an autopsy cohort of neuropathologically verified non-demented controls and subjects with Alzheimer's disease (AD) resident in nursing homes in the Oslo area. AD was associated with a significantly increased frequency of the Apo E epsilon 4 allele; the frequency of the epsilon 2 and epsilon 3 alleles was lower in AD but not significantly so. Age at death in the control group and the AD group did not differ significantly; neither did age at death nor age at onset of dementia in AD vary according to Apo E genotype, though tendencies towards an earlier age at death was seen in individuals with epsilon 4/4 and earlier age at onset dementia in the presence of an epsilon 4 allele and a later age of onset the presence of an epsilon 3 allele were seen. Possession of an epsilon 2 allele had no effect on age at onset of dementia or age at death. Among the possible genotypes there was a trend towards a progression of earliest onset epsilon 4/4, epsilon 2/4, epsilon 3/4, epsilon 3/3, epsilon 2/3 latest onset of dementia and longest duration epsilon 2/4, epsilon 4/4, epsilon 3/4, epsilon 3/3, epsilon 2/3 to shortest duration of dementia.

Content of brain aluminum is not elevated in Alzheimer disease.

Several studies have reported that the bulk aluminum (Al) concentration is increased in the brain in Alzheimer disease (AD), while other studies have failed to demonstrate an increase. Most of these investigations have had one or more methodological deficiencies, including lack of adequate neuropathological assessment; failure to age-match the control samples; small sample sizes, lacking statistical power; and geographical heterogeneity in the AD and control populations. The present population-based study of 92 clinically and histopathologically diagnosed AD patients and normal elderly nursing home residents was designed to avoid these potential biases. When a subsample of AD cases with the most severe brain pathology was compared with controls having no or minimal pathology, no statistically significant differences were found in the bulk aluminum concentration measured by graphite furnace atomic absorption spectrometry in frontal cortex (1.8 +/- 0.7 vs. 1.7 +/- 0.7 micrograms/g dry wt), temporal cortex (1.4 +/- 0.3 vs. 1.5 +/- 0.5 micrograms/g dry wt), liver (2.0 +/- 1.3 vs. 2.0 +/- 1.2 micrograms/g dry wt), or head of femur (2.4 +/- 1.6 vs. 2.2 +/- 1.0 micrograms/g ash wt). Within the whole series of 92 cases, there was no difference in the bulk aluminum concentration of the frontal cortex between individuals diagnosed as definite, probable, and possible cases of AD using the CERAD (Consortium to Establish a Registry for Alzheimer's Disease) criteria. The density of senile plaques and neurofibrillary tangles in frontal and temporal cortex showed no correlation with the bulk aluminum concentration. Logistic regression analyses, which controlled for age and sex, did not influence outcome for any of the comparisons. The data show conclusively that in AD, bulk aluminum concentration is not increased in two cortical brain regions that are selectively vulnerable to the neuropathological changes associated with this disorder.

Effect of apolipoprotein E genotype on Alzheimer's disease neuropathology in a cohort of elderly Norwegians.

A cohort of elderly Norwegians dying in nursing homes in the Oslo region have been genotyped for the Apolipoprotein E (ApoE) gene. Alzheimer's disease (AD) cortical neuropathology and clinical evidence of dementia were used to assign cases without evidence of other confounding neuropathology. Senile plaque (SP) and neurofibrillary tangle (NFT) densities in frontal, temporal and parietal cortex were then correlated with ApoE genotype to determine any relationship between ApoE genotype and AD pathology. Comparisons with ApoE epsilon 3, epsilon 4 and epsilon 2 allele dosage failed to show any significant effect on cortical SP densities in any cortical area. NFT densities were increased by epsilon 4 allele dosage in the frontal cortex but not in other cortical regions. A reduction was seen in cortical NFT densities with epsilon 2 allele, though again this was not consistently significant in any of the groups. The epsilon 3 allele failed to show any consistent effect on cortical NFT densities. Assessment by individual genotypes showed epsilon 2/3 < epsilon 2/4 < epsilon 3/3 < epsilon 3/4 < epsilon 4/4 which had highest cortical NFT densities in all areas. By genotype, SP densities were generally of the order epsilon 2/4 < epsilon 2/3 < epsilon 3/3 < epsilon 4/4 < epsilon 3/4 though in none of the groups was this significant. Duration of disease showed no consistent effect on neuropathological burden. ApoE genotype may have an effect on determining whether individuals suffer from AD and the age at onset of disease but may only have a minimal effect on pathology burden.

Changing incidence of HIV-induced brain lesions in Oslo, 1983-1994: effects of zidovudine treatment.

OBJECTIVE: To investigate the relation between HIV-induced brain lesions, zidovudine (ZDV) treatment and survival length in a well-defined population of HIV-positive patients. METHODS AND PATIENTS: Ulleval Hospital has the responsibility for treating all AIDS patients from the city of Oslo except haemophiliac patients. The patient population in this autopsy study comprised all adult AIDS patients in Oslo who were treated at our hospital and died during 1983-1994 (n = 171). This represents 86% of all adult AIDS patients from Oslo who died during the same period. Full autopsy, including neuropathological examination of the brain and spinal cord, was performed on 128 (75%) of those who died. RESULTS: No significant differences were found between autopsy and non-autopsy cases with regard to sex, age, risk groups, survival length or ZDV treatment. In the autopsy material, multinucleated giant cells (MGC) in brain tissue were found in 29 cases and diffuse damage of white matter in 52 cases. Analysis shows that ZDV (600 mg per day) reduced the incidence of these brain lesions, but only if continued until death. A second finding was an increased incidence of HIV-induced brain lesions for those with long-term survival. Together these observations may explain a substantial part of the time-trend in the incidence of MGC in Oslo. MGC were frequent (40%) during the first years of the epidemic, although survival length was short in this period. The incidence fell markedly around the time ZDV was introduced and later remained low in those using ZDV until death. The incidence of MGC has, however, increased during the later years, the new cases mainly occurring in patients who had discontinued ZDV use. CONCLUSION: If continued until death, ZDV can reduce the incidence of HIV-induced brain lesions in AIDS patients. When ZDV treatment is terminated a rapid increase occurs in the incidence of HIV encephalitis.

Neuropathological diagnoses in elderly patients in Oslo: Alzheimer's disease, Lewy body disease, vascular lesions.

Neuropathological changes in elderly residents of Oslo, Norway were characterised with respect to the cerebral substrates of dementia. Ninety-two brains were examined, representing 41% of all deaths occurring in 10 nursing homes during a 9-month period. The autopsy cohort showed a similar mean age (85 years) and sex ratio (73% female) and proportion of demented patients (75%) compared to all the patients resident in these homes who died during the same period. Clinical data was compiled retrospectively. Diagnosis was made using the CERAD protocol, and criteria for the diagnosis of Lewy body dementia. Lewy body formation was present in 20% and cerebral infarction in 21% of patients. In the demented group (69 patients) 90% fulfilled CERAD criteria for definite or probable Alzheimer's disease. Eight demented cases had absent neocortical neurofibrillary tangles and 6 other cases showed Lewy body dementia (9% of demented patients). A further 8 of these demented cases had brain stem Lewy bodies with only minimal cortical involvement. Thirteen cases (19% of the sample) had cerebral infarcts but these were considered to be clinically significant in only 4 (6%). In the non-demented patients (23) 4 patients had brain stem Lewy bodies and 6 had cerebral infarcts. Despite inclusion criteria biased towards the collection of Alzheimer's disease and normal patients, both Lewy body dementia (7%) and cerebral infarcts contributing to dementia (6%) were frequent.

[The Liland case. Medicolegal aspects]. / Lilandsaken. De rettsmedisinske erklaeringer.

In 1970 a 39 year-old man was sentenced to lifelong imprisonment (21 years) plus ten years of preventive detention for having killed two persons with an axe on December 22, 1969. The sentence was based upon circumstantial evidence and medicolegal reports which suggested that these persons had been killed on December 22 or early December 23. The accused had an unquestionable alibi for December 23. Re-examination of the medicolegal reports 24 years after the verdict demonstrated that the killings could not have taken place on December 22. Therefore, in a new trial on November 21, 1994, the accused was fully acquitted. Measures by which similar medicolegal errors can be prevented are discussed.

Neurotoxicity of water-soluble contrast media.

A review is given of the development of the water-soluble contrast media (CM) with particular attention to the frquency of neurological complications. A remarkable improvement was achieved following the introduction of the nonionic agent metrizamide in 1974, and a further decrease in neurotoxicity was obtained with the newer nonionic monomers, which have multlple hydroxyl groups included at different sites of the molecule. Theoretical considerations and experimental studies suggest that the neurotoxicity of the new nonionic dimeric agents shuold be at least within the low range seen with the monomeric ones, but further experience is needed before definite conclusions can be drawn in this respect. The mechanisms responsible for the neurological complications seen with CM are unknown but certain critical groups on the CM molecules are known. Several animal models have been developd, which may help predict the degree of neurotoxicity.

Early entry of plasma proteins into damaged neurons in brain infarcts. An immunohistochemical study on experimental animals.

Entry of plasma proteins into damaged neurons has previously been demonstrated in various pathological conditions, but little is known about brain infarcts in this respect. In the present study, focal ischemic lesions were produced in rats by permanent occlusion of the middle cerebral artery (MCA). The animals were killed from 1 to 48 h postlesion. Leakage of plasma proteins across the blood-brain barrier into the infarcted area was visualized with immunostaining 2-3 h after the occlusion. This is earlier than in most previous reports. Entry of plasma proteins into ischemic neurons was seen 3 h after permanent occlusion of the MCA, while reliable changes were not seen until 12-24 h in sections stained with hematoxylin and eosin (H & E). Ischemic neurons stained for plasma proteins irrespective of their morphological appearance. Even cells that appeared normal with H & E staining were positively labeled. The technique may be used to diagnose very early ischemic lesions.

[Slow replenishment of carnitine level after long-term treatment with pivampicillin/pivmecillinam]. / Langsom gjenoppbygging av karnitinnivå etter langtidsbehandling med pivampicillin/pivmecillinam.

Long-term treatment with pivampicillin and pivmecillinam for 6-24 months in five adults and one child reduced the total serum carnitine concentrations to 3.7-14.0 mumol/l (reference value 25-66 mumol/l). The muscle carnitine was reduced to 0.3-0.7 mumol/g wet weight (reference value 3-5 mumol/g) in two cases. All patients had asthenia and muscle symptoms with weakness and pain. One showed signs of carnitine depletion in the liver, with increased secretion of dicarboxylic acids (C6, C8, C10) in urine and limited ketone body formation during prolonged fasting (32 hours). The serum carnitine increased slowly after cessation of therapy and reached normal concentrations after 6-12 months. All symptoms caused by carnitine depletion disappeared after the serum carnitine reached 20 mumol/l. This was achieved on a normal diet without carnitine supplement.

Does granulocyte depletion protect against ischaemic muscle necrosis?

Male Wistar rats were submitted to complete hindlimb ischaemia for 4.5 h. Six animals were injected with antineutrophilic serum (ANS) pre- and post-operatively, and eight animals received normal sheep serum (NSS). The rats were killed 72 h after the insult, and the areas of necrosis in the anterior tibial muscles were measured morphometrically on histological slides. In the NSS group there was a considerable postoperative increase in polymorphonuclear leukocytes (PMNLs) in peripheral blood, while treatment with ANS resulted in total depletion of PMNLs during ischaemia and early reperfusion. There was 31% necrosis in the NSS group and 28% necrosis in the ANS group. This difference was not significant (p = 0.7). It is concluded that depletion of PMNLs had no effect on the infarct size in the anterior tibial muscle after 4.5 h of complete hindlimb ischaemia and 72 h of reperfusion.

Pentoxifylline reduces skeletal muscle necrosis after acute hindlimb ischemia in rats.

Complete ischemia in the left hindlimb was maintained for 3.5 h in 16 rats randomized into two equal groups. One group served as control, the other was treated with pentoxifylline before, during and after the ischemic period. The animals were killed 72 h later, and the anterior tibial muscles were prepared for histological investigation. The most severely damaged muscles had a central core with complete necrosis of all muscle fibers, disintegrated capillaries and no macrophage infiltration. This zone, which was called the area of no resorption, was surrounded by an area of incomplete necrosis with partly resorbed muscle fibers, intact capillaries and marked macrophage infiltration. The muscles were completely intact only in a narrow subfascial zone. The total areas of necrosis and the areas of no resorption were measured as percent of the cross-sectional area of each muscle. There was extensive necrosis in both groups. The infarcted area was 93% in the control group and 89% in the treated group (NS). The corresponding areas of no resorption were 23 and 6% (p = 0.01). The study indicates that pentoxifylline has some protective effect on ischemic muscle damage. However, this effect is marginal compared to moderate hypothermic treatment.

Neuronal uptake of plasma proteins after transient cerebral ischemia/hypoxia. Immunohistochemical studies on experimental animals and human brains.

Rapid uptake of plasma proteins into damaged neurons has been demonstrated previously after lesions which cause early breakdown of the blood-brain barrier (BBB). The present study was undertaken to see whether a similar uptake occurred after hypoxic/ischemic episodes in men and experimental animals. Forebrain ischemia was produced in rats by a combination of carotid clamping and hypotension for 15 min, followed by recirculation for 6 h, 24 h, 48 h and 5 d. Paraffin sections from the brains were incubated with antiserum against albumin, and parallel sections were stained with hematoxylin and eosin (H & E). Breakdown of the BBB with extravasation of albumin was seen after 6 h in the lateral reticular nucleus of the thalamus, the dorsolateral striatum, and in restricted areas of the cerebral cortex. Uptake of albumin into damaged neurons was seen in the same structures, and partly before reliable changes were observed in routinely stained sections. With longer survival periods, the staining of the neuropil became stronger and more neurons in the damaged areas were positively labeled. After 48 h and 5 d many neurons in the hippocampal sector CA1 had also taken up plasma proteins. A similar uptake of plasma proteins into damaged neurons was seen in brains from patients with histological evidence of hypoxic injury. Even the small leakage of proteins that occurs after hypoxic/ischemic lesions is thus sufficient to give a definite immunostaining of damaged neurons.

Slow replenishment of carnitine deficiency after cessation of long-term treatment with pivaloyl-containing antibiotics.

Long-term treatment with pivampicillin and pivmecillinam for 6-24 months in five adults and one child reduced the total serum carnitine concentration to 3.7-14 mumol/l (reference value: 25-66 mumol/l). Muscle carnitine was reduced to 0.3-0.7 mumol/g wet weight (reference value: 3-5 mumol/g) in two cases. All patients had muscle symptoms with weakness, asthenia and pains. One showed signs of carnitine depletion in the liver with increased secretion of dicarboxylic acids (C6, C8, C10) in urine and limited ketone body formation during prolonged fasting. Serum carnitine increased slowly after cessation of therapy and reached normal concentrations after 6-12 months. All symptoms caused by carnitine depletion disappeared. This was achieved on a normal diet without carnitine supplementation.

Distinction between artefactually shrunken and truly degenerated 'dark' neurons by in situ fixation with microwave irradiation.

Dark, shrunken neurons frequently occur as artefacts in immersion fixed tissue. Perfusion fixation will prevent artefacts of this type. However, morphologically identical neurons have been described as truly degenerated cells in perfusion fixed brains in various pathological conditions. Since adequate perfusion is difficult to obtain in some pathological conditions, the question still remains whether the dark neurons found in some of these situations are true in vivo phenomena or artefacts caused by inadequate fixation. In the present study rat brains with cryogenic lesions were fixed in situ by microwave irradiation. With this method no artefactually changed dark neurons were observed in the normal parts of the brains. In the cryogenic lesions, however, a narrow rim of dark, shrunken neurons occurred adjacent to the normal cortex. This zone was identical to that observed in perfusion fixed tissue. Since inadequate fixation due to uneven perfusion of the damaged tissue is prevented with this method, we suggest that the neuronal changes represent true in vivo phenomena. Fixation with microwave irradiation can thus be used to differentiate between artefactually changed and truly degenerated dark neurons in various pathological conditions.

Perfusion in the anterior tibial muscle measured by laser Doppler flowmetry after graded periods of hindlimb ischemia in rats.

In the present study laser Doppler flowmetry was used to evaluate skeletal muscle perfusion before and after acute hindlimb ischemia in rats. Three laser Doppler microprobes were placed in the anterior tibial muscles of both legs. Six animals were used as non-ischemic controls and 22 animals were subjected to complete ischemia in the left hindlimb for 0.5 to 4.5 hr. By calculating a perfusion index based on recordings from the left and the right leg, a continuous measurement of relative perfusion could be performed. Reactive hyperemia was demonstrated after ischemia of shorter than 2 hr duration. After longer occlusion periods, the reperfusion decreased with increasing duration of ischemia. It is concluded that the use of laser Doppler microprobes offers possibility for continuous evaluation of relative microvascular blood flow in normal and postischemic skeletal muscle, and that the technique would be useful in experimental studies concerning ischemia-reperfusion injury of skeletal muscles.

Local hypothermia during ischemia or reperfusion in skeletal muscles.

A modified rat hindlimb tourniquet model was used to measure postischemic muscle necrosis. The effect of moderate local hypothermia to 20 degrees C during ischemia and reperfusion was investigated. Eighteen animals were kept in an incubator at 27 degrees C, and complete circulatory arrest was maintained for 3.5 h before release of the vascular occlusion. After survival for 72 h the degree of necrosis in the anterior tibial muscles was measured morphometrically on histological slides. Areas of necrosis with intact capillary structure and resorption of muscle fibers, and areas without resorption and capillary disintegration were measured separately. Three experimental groups (six animals in each) were included in the series. In the first group local cooling to 20 degrees C was performed during the initial 1.5 h of ischemia. The second group was cooled for 1.5 h during the initial phase of reperfusion. The animals in the third group served as controls without cooling. The total areas of necrosis in the three groups were 0, 90 and 90%, and the areas of no-resorption 0, 23 and 39%, respectively. Cooling during ischemia thus had a marked effect, while no significant differences were found between the control group and the group cooled during reperfusion. The study shows that moderate cooling during initial ischemia protects effectively against postischemic muscle necrosis, while cooling during reperfusion has no such effect.

Histological studies on postischemic rat skeletal muscles. With emphasis on the time of leukocyte invasion.

Several studies have suggested that the damage after ischemic injuries increases during reperfusion and that this reperfusion damage is mediated through granulocytes which invade the damaged area. The present study was undertaken to test these hypotheses by histological investigations of the anterior tibial muscles in a rat hind limb tourniquet model after 4.5 h of complete ischemia and graded periods of reperfusion. Uptake of albumin into damaged muscle fibers was demonstrated immunohistochemically and used to determine the extent of the ischemic lesions. The size of the lesions was measured morphometrically on immunostained sections from paraffin-embedded material. Granulocytes in and outside the capillaries were counted on slides from historesin-embedded material. Generally, there was a compact central area in the muscles which showed necrosis of all fibers and many capillaries, little interstitial edema, and little or no invasion of granulocytes or macrophages. The central core did not increase in size during the postoperative period. Outside this are there was a zone of partial tissue destruction with quite marked interstitial edema. This zone also remained unchanged in size but it is uncertain whether the number of necrotic fibers increased with time. There was an increasing invasion of granulocytes in this outer zone from 5 to 24 h after release of the occlusion and monocyte/macrophage invasion was seen from 48 h. Outside this zone there was a subfacial zone with normal muscle fibers in all cases. It is concluded that the central area of complete tissue destruction was present at the time of release of the occlusion and did not increase in size during the further postoperative course. There was no indication that granulocytes participated in this damage. The extent of the partially damaged area also remained unchanged during the postoperative course. It is conceivable that granulocytes aggravated the lesions by increasing the number of necrotic fibers in this peripheral area but not before 5 h after release of the occlusion. This is later than described in previous studies.