Effects of methotrexate on astrocytes in primary culture: light and electron microscopic studies.

Recent studies on the animal model suggest that astrocytes may be a primary target for methotrexate (MTX) toxicity. To establish whether the astroglial alterations are due to a direct toxic effect of the drug, we studied the morphologic alterations, mitotic index, viability and growth rate of astrocytes in primary culture after exposure to varying concentrations of MTX in the absence or presence of dibutyryl cyclic AMP (dBcAMP). Dense bodies and cellular debris were noted by light and electron microscopy, and became more prominent with increasing doses and greater frequency of treatment. Degenerating cells and areas of necrosis were seen at higher concentrations. These changes became less conspicuous when MTX was given concurrently with dBcAMP. Large reactive-like astrocytes were also seen after MTX administration both in the absence or presence of dBcAMP. Mitotic rate inhibition was noted at all concentrations but was not dose-related. Cell viability was reduced and remained low up to 48 h after withdrawal of MTX and correlated well with drug concentration, although growth rate did not vary significantly from the control. Our findings show that pure populations of astrocytes can be adversely affected by MTX especially in the absence of bBcAMP, while also causing reactive-like changes in some cells. This report provides further evidence that astrocytes may be a primary target for MTX toxicity and suggests that the gliosis seen in MTX encephalopathy may in part be related to MTX-induced astrocytic injury.

Morphologic alterations in rat brain following systemic and intraventricular methotrexate injection: light and electron microscopic studies.

To determine the morphological substrate of acute methotrexate (MTX) encephalopathy, light and electron microscopic studies were performed on rat brains after short-term intraperitoneal (IP) and intraventricular (IV) injections of MTX. In both models, Alzheimer type II astrocytosis was the initial and major pathologic alteration seen by light microscopy. The neurons, oligodendrocytes, myelin and endothelial cells were relatively spared. Ultrastructural studies showed pleomorphism and condensation of mitochondria, membrane-bound vacuoles, prominent stacks of sparsely granular, rough endoplasmic reticulum and progressive hydropic swelling of astrocytic perikarya and their processes. The astroglial alterations were reversible after cessation of the drug but persisted for a longer time with repeated IP administration. Gastrointestinal complications and overall mortality were also greater with higher doses and increasing frequency of IP MTX injection. White matter necrosis was noted only after IV injection of high-dose MTX. The neuropathologic changes of MTX leukoencephalopathy can be replicated in an animal model by IV injection of the drug. The reversibility of the changes that were seen following IP administration correlates with the transient neurologic deficits observed in some patients after high-dose systemic MTX therapy. The initially selective astroglial effect suggests that astrocytes might be a target for MTX toxicity, although other central nervous system components may also be adversely affected by the drug.

Prophylactic indomethacin for prevention of intraventricular hemorrhage in premature infants.

The impact of early prophylactic use of intravenous indomethacin on the incidence and severity of periventricular-intraventricular hemorrhage and patent ductus arteriosus in 199 oxygen-requiring premature infants (less than or equal to 1300 g birth weight) was prospectively investigated. The trial was controlled, the infants were randomized, and the investigators were unaware of the group assignments. Patients with minimal (grade I) or no periventricular-intraventricular hemorrhage determined by prestudy echoencephalography were randomized within two birth weight subgroups (500 to 899 and 900 to 1300 g) to receive either prophylactic indomethacin (n = 99) or an equal volume of saline-vehicle placebo (n = 100). The first dose (0.2 mg/kg) was given within 12 hours of delivery and two subsequent doses (0.1 mg/kg) were administered at 12 hourly intervals. Prophylactic indomethacin significantly reduced the incidence of grades II to IV periventricular-intraventricular hemorrhage. Intraventricular hemorrhage was half as common in infants given prophylactic indomethacin as in control infants (23% v 46%, P less than .002). The reduction was manifested in both birth weight subgroups. Results of this study also confirmed a lower incidence of clinically significant patent ductus arteriosus in infants who received prophylactic indomethacin in contrast to those who received placebo (11% v 42%, P less than .001). No significant differences were found between treatment and control groups in the duration of oxygen therapy, mechanical ventilation, or hospitalization or in the incidence of pneumothorax, chronic lung disease, sepsis, necrotizing enterocolitis, retinopathy of prematurity, or death. Early prophylactic indomethacin initiated within 12 hours of delivery is effective in reducing the incidence of intraventricular hemorrhage as well as clinically significant patent ductus arteriosus in very low birth weight premature infants.

Malignant angioendotheliomatosis--a true lymphoma: a case of intravascular malignant lymphomatosis studied by southern blot hybridization analysis.

Malignant angioendotheliomatosis is a rare, systemic, usually fatal disease characterized by a massive proliferation of large, bizarre-looking mononuclear cells within small and medium-sized blood vessels. The histogenesis of the neoplastic cells has been the subject of long-standing controversy since the disease's initial description. Early investigators concluded that the entity represented a neoplasm of endothelial cells, but recently others have suggested that it is of lymphoid origin. We studied a case of malignant angioendotheliomatosis by Southern blot hybridization analysis which showed clonal rearrangements of the immunoglobulin heavy-chain gene strongly suggesting a B-lymphocyte origin. Our results provide additional evidence that malignant angioendotheliomatosis is an intravascular malignant lymphomatosis.

Homozygous protein C deficiency in a newborn. Clinicopathologic correlation.

A rare case of homozygous protein C deficiency occurred in a newborn. The patient presented with purpura fulminans in the first few hours after birth and showed multiple hemorrhagic lesions on computed tomography of the brain at 5 days of age. Neurologic symptoms developed at two weeks and the patient died at 37 weeks. His protein C level was less than 5%. Autopsy revealed thrombosis of the dural sinuses, multiple cortical infarcts, intraparenchymal hemorrhages, and hydrocephalus. The pathologic findings are correlated with the neurologic deficits and previously documented cases are reviewed.

Encephalomyelitis associated with leukemia in a child.

A case of an encephalomyelitis in a child with acute lymphoblastic leukemia is reported. The patient was a 5-year-old boy who developed seizures, progressive confusion, and coma after radiation and intrathecal methotrexate therapy. Computed tomography (CT) of the brain showed bilateral hypodensities in the posterior parietal and temporal regions. At autopsy, perivascular inflammation, microglial nodules without intranuclear viral inclusions, and bilateral necrosis of the temporoparietal and hippocampal regions were seen in the brain and spinal cord. Paraneoplastic encephalomyelitis is generally recognized in adult patients with underlying malignancy but, to our knowledge, has not been reported in children with leukemia. This report should alert the clinicians to an entity that must be included in the differential diagnosis of leukemic children with progressive neurologic disorder.

Effects of lactic acid on astrocytes in primary culture.

Excessive tissue lactic acidosis is considered to be detrimental to the central nervous system (CNS) and may adversely affect recovery from anoxia, ischemia, trauma and epilepsy. Since astrocytes are believed to play a role in pH regulation in the CNS, we studied the effect of this acid on primary astrocyte cultures. Cells exposed to lactic acid showed chromatin clumping, an increase of lipid and dense bodies, a loss of polyribosomal clusters, slightly increased cytoplasmic lucency, swollen mitochondria and tangled intermediate filaments. These alterations progressed with lower pH and longer exposure. Irreversible changes occurred one to two hours after exposure at pH 6; after 30 to 60 minutes (min) at pH 5.5 and after ten to 30 min at pH 5. Comparable results were obtained with the use of other weak acids indicating that the observed changes were due to increased hydrogen ion concentration rather than secondary to lactate per se. Additionally, various concentrations of lactic acid adjusted to identical pH produced similar morphologic alterations. Thus, while lactic acid caused marked and at times irreversible alterations in astrocytes, severe and prolonged acidosis was required to produce such injurious effects. This relative resistance of astrocytes to acidosis is in keeping with their potential role in pH regulation in brain.

Progressive cytomegalovirus encephalopathy following congenital infection in an infant with acquired immunodeficiency syndrome.

Congenital central nervous system infection with cytomegalovirus (CMV) usually results in a nonprogressive encephalopathy. Ninety percent of patients with clinically apparent infections at birth have a permanent neurological disability. It has been suggested that some infants may have persistent infection manifested by progressive encephalopathy during infancy. In the present case, clinical and pathological findings suggest the reactivation of a prior intrauterine CMV infection in a child with human T-lymphotrophic virus type III (HTLV-III) infection. The presence of HTLV-III may have reduced the immune surveillance of this infant, allowing the CMV to reactivate.

Spinal cord tumors presenting with neural tube defects.

Two primary lumbosacral tumors arising at the site of neural tube defects are presented. One was a teratoma diagnosed in an infant with myelomeningocele. The other was an ependymoma that developed in an adult with meningocele. It is postulated that these cases represent a neoplastic transformation of heterotopic primordial elements that have been incorporated within the defect, supporting the view that overgrowth of neural tissue may be the result rather than the cause of neural tube deformity. Such rare occurrences may be due to interaction between intrauterine teratogenic factors and familial predisposition in affected patients.

Effect of cyclic AMP on ammonia-induced alterations in primary astrocyte cultures.

Current evidence suggests that astrocytes may be the target of ammonia toxicity. Consistent with this view are recent investigations which have shown morphologic alterations in primary astrocyte cultures following exposure to ammonia. In the present study, these alterations became severely aggravated when the cultures were not grown or maintained in dibutyryl cyclic adenosine monophosphate (AMP). Cyclic AMP analogues and agents that increase intracellular cyclic AMP levels significantly inhibited the toxic effects of ammonia. The exact mechanism responsible for this apparent protective effect of cyclic AMP on ammonia-treated astrocytes is not known. The possible means by which cyclic AMP may serve to ameliorate ammonia-induced toxicity are discussed.

Fatal metabolic acidosis, hyperglycemia, and coma after steroid therapy for Kearns-Sayre syndrome.

Two adolescent boys with Kearns-Sayre syndrome (progressive external ophthalmoplegia, heart block, elevated CSF protein, and ragged-red muscle fibers) developed lethargy, increasing somnolence, polydipsia, polyphagia, and polyuria after a brief course of steroid therapy. Both had hyperglycemia and acidosis. Nonketotic, lactic acidosis was present in one and ketosis in the other. Severe respiratory failure developed, and both patients died. Postmortem revealed fatty infiltration of the pancreas in addition to a diffuse spongiform encephalopathy.

Reactivation of congenital cytomegalic inclusion disease in an infant with HTLV-III associated immunodeficiency: a CT-pathologic correlation.

Serial CT findings in an infant with HTLV-III infection and cytomegalic inclusion virus encephalitis are presented.

Morphologic effects of ammonia on primary astrocyte cultures. II. Electron microscopic studies.

Light microscopic studies of primary astrocyte cultures following exposure to ammonia have shown several alterations. To determine the nature and significance of these changes, electron microscopic studies were performed. Ultrastructural changes consisted of proliferation, pleomorphism and swelling of mitochondria, condensation of the mitochondrial matrix, cytoplasmic lucency and vacuolization, disaggregation of polyribosomal clusters, an initial increase followed by degranulation of rough endoplasmic reticulum, proliferation of smooth endoplasmic reticulum, an accumulation of dense bodies and a loss of intermediate glial filaments. The early alterations appeared reactive and perhaps reflected ammonia detoxification. Some changes were degenerative and support the view that ammonia exerts a direct toxic effect on astrocytes. It is postulated that these changes may interfere with critical astroglial functions and thereby play a key role in the neurologic dysfunction seen in hyperammonemia.

Morphologic effects of ammonia on primary astrocyte cultures. I. Light microscopic studies.

To evaluate the astrocytic alterations commonly seen in hepatic encephalopathy and other hyperammonemic states, primary astrocyte cultures derived from neonatal rats were exposed to varying concentrations of ammonia for one to ten days. Ammonia-treated cultures initially showed an increase in basophilia, prominent cytoplasmic processes and increased cytoplasmic granularity and vacuolization. Nucleoli were increased in size and there was an increase in nucleolar/nuclear ratio. Later, fragmentation and loss of cytoplasmic processes, formation of dense bodies and frank cellular disintegration were noted. The changes were proportional to the concentration and duration of ammonia treatment. Our studies show that ammonia is capable of directly causing morphologic alterations in astrocytes. We believe that the use of primary cultures provides a means of exploring the precise role of astrocytes in hyperammonemic states.

Cytomegalovirus. Induced demyelination associated with acquired immune deficiency syndrome.

We observed three cases of cytomegalovirus (CMV) infection of the nervous system in association with acquired immune deficiency syndrome. The first patient had demyelinating lesions in the spinal cord with relative preservation of axis cylinders. The second patient had a discrete focus of demyelination in the hypothalamic region associated with well-preserved axons and intact neurons. Microglial nodules were absent in both patients. The third patient had subacute encephalomyelitis principally encephalomyelitis principally characterized by microglial nodule formation in the brain and spinal cord, in addition to necrotizing lesions in the thoracic cord and segmental demyelination in the anterior spinal nerve roots. Perivascular lymphocytic infiltration was minimal and was noted mainly within the peripheral nerves. Typical cytomegalic intranuclear inclusion bodies were seen in the nervous tissue of these three patients who all had evidence of disseminated CMV infection. It is possible that these patients had virus-induced demyelination in the face of altered immunoregulation.

Progressive multifocal leukoencephalopathy in a patient with acquired immune deficiency syndrome.

Progressive multifocal leukoencephalopathy (PML) occurred in a heterosexual Haitian man with acquired immune deficiency syndrome (AIDS). The patient initially had focal neurologic signs and nonenhancing lesions on a computed tomographic scan. Although PML is rare, it should be included in the differential diagnosis of opportunistic infections associated with AIDS. Brain biopsy is suggested in patients suspected of having PML who might benefit from antiviral therapy.

Extramedullary hematopoiesis in a malignant meningioma.

An unusual case of malignant intracranial meningioma is presented. The operative management was complicated by the abrupt development of fulminant brain edema and herniation. The tumor contained areas of extramedullary hematopoiesis, a finding not previously reported. Various pathogenetic mechanisms involved in intradural extramedullary hematopoiesis are discussed.

Leukoencephalopathy associated with extensive burns.

Unusual neuropathological changes were observed in two cases following extensive burns. These consisted of perivascular areas of demyelination distributed symmetrically in the brain and affecting the white matter predominantly. One case in addition had widespread petecchial and ring haemorrhages characteristic of brain purpura. Both patients sustained second and third degree burns in greater than 50% of the body surface area, developed metabolic acidosis, sepsis, disturbance in consciousness and multiple episodes of cardiorespiratory arrest prior to death. A toxic metabolic state related to a burn toxin released from the damaged tissue or from bacterial action to the tissue in addition to low platelet level is proposed as the major pathogenetic factor in the development of the neurological symptoms and the patients' demise.