Suni G. Allen

Hypoxic neuronal necrosis: Protein synthesis-independent activation of a cell death program

Hypoxic necrosis of dentate gyrus neurons in primary culture required the activation of an orderly cell death program independent of protein synthesis. Early mitochondrial swelling and loss of the mitochondrial membrane potential were accompanied by release of cytochrome c and followed by caspase-9-dependent activation of caspase-3. Caspase-3 and -9 inhibitors reduced neuronal necrosis. Calcium directly induced cytochrome c release from isolated mitochondria. Hypoxic neuronal necrosis may be an active process in which the direct effect of hypoxia on mitochondria may lead to the final common pathway of caspase-3-mediated neuronal death.

Vulnerability of postnatal hippocampal neurons to seizures varies regionally with their maturational stage.

The mechanism of status epilepticus-induced neuronal death in the immature brain is not fully understood. In the present study, we examined the contribution of caspases in our lithium-pilocarpine model of status epilepticus in 14 days old rat pups. In CA1, upregulation of caspase-8, but not caspase-9, preceded caspase-3 activation in morphologically necrotic cells. Pretreatment with a pan-caspase inhibitor provided neuroprotection, showing that caspase activation was not an epiphenomenon but contributed to neuronal necrosis. By contrast, upregulation of active caspase-9 and caspase-3, but not caspase-8, was detected in apoptotic dentate gyrus neurons, which were immunoreactive for doublecortin and calbindin-negative, two features of immature neurons. These results suggest that, in cells which are aligned in series as parts of the same excitatory hippocampal circuit, the same seizures induce neuronal death through different mechanisms. The regional level of neuronal maturity may be a determining factor in the execution of a specific death program.

Status Epilepticus Triggers Caspase-3 Activation and Necrosis in the Immature Rat Brain

Summary:  The mode and mechanism of neuronal death induced by status epilepticus (SE) in the immature brain have not been fully characterized. In this study, we analyzed the contribution of neuronal necrosis and caspase‐3 activation to CA1 damage following lithium‐pilocarpine SE in P14 rat pups. By electron microscopy, many CA1 neurons displayed evidence of early necrosis 6 hours following SE, and the full ultrastructural features of necrosis at 24–72 hours. Caspase‐3 was activated in injured (acidophilic) neurons 24 hours following SE, raising the possibility that they died by caspase‐dependent “programmed” necrosis.

ALPHA‐1 ANTITRYPSIN DEFICIENCY IN A PATIENT WITH WIDESPREAD PRURIGO NODULARIS

Skin lesions associated with alpha1‐antitrypsin deficiency are becoming better defined and understood. Deficiency in this major antiproteinase, which neutralizes multiple proteolytic enzymes ranging from collagenases and elastases to trypsin and chymotrypsin, thus results in significant tissue autodigestion. This anti‐proteinase is secreted by activated lymphocytes and macrophages, suggesting the existence of homeostasis which titrates the release of proteolytic enzymes by these cells, and the adequate neutralization of these proteases in order to prevent excessive tissue autodigestion each time these inflammatory cells are activated. We report a patient with alpha1‐antitrypsin deficiency who, following insect bites and cellulitis developed widespread itching and scratching, leading to widespread lesions of prurigo nodularis. The colonization of his multiple skin lesions with Staphylococcus aureus and the release of potent T cell mitogens, such as Protein A and enterotoxin A from the bacterial cell membrane may have resulted in the release of additional proteolytic enzymes by the activated lymphocytes and macrophages, without the concomitant secretion of alpha1‐antitrypsin with subsequent aggravation of his pruritus. These concepts are supported by electron microscopic evidence of excessive tissue autodigestion, and by immunocytochemical data identifying the presence of T helper and T cytotoxic/suppressor lymphocytes as well as macrophages within the upper dermis.

Involvement of Luminal Bacteria, Heat Shock Protein 60, Macrophages and γδ T Cells in Dextran Sulfate Sodium-Induced Colitis in Rats

The in vivo immunological events in dextran sulfate sodium (DSS) -induced colitis were evaluated. Rats were fed water (control) or 5% DSS. Colonic sections were assessed by light microscopy, Gram stain, immunohistochemistry, and electron microscopy. A progressive decline in number and increase in fragmentation of bacteria in the colonic lumen was observed over time. Luminal bacteria were the first to show heat shock protein 60 (HSP60) staining (day 3). Macrophages in close proximity to these bacteria were next to show such staining (day 6), and finally the damaged epithelial cells when colitis became severe (day 15). Ultrastructural assessment showed cell–cell contact interactions between macrophages and dendritic γδ T cells. An increase in the number of γδ T cells and ED1-positive macrophages in the affected colonic tissue over time was documented. These results suggest colonic bacteria, host macrophages, and γδ T cells play specific roles in the immunological reactions in DSS-induced colitis, possibly via an HSP60-mediated mechanism.

CONTRIBUTION OF A MITOCHONDRIAL PATHWAY TO EXCITOTOXIC NEURONAL NECROSIS

It is traditionally thought that excitotoxic necrosis is a passive mechanism that does not require the activation of a cell death program. In this study, we examined the contribution of the cytochrome c‐dependent mitochondrial death pathway to excitotoxic neuronal necrosis, induced by exposing cultured cortical neurons to 1 mM glutamate for 6 hr and blocked by the NMDA antagonist, dizocilpine. Glutamate treatment induced early cytochrome c release, followed by activation of caspase‐9 and caspase‐3. Preincubation with the caspase‐9 inhibitor z‐LEHD‐fmk, the caspase‐3 inhibitor z‐DEVD‐fmk, or the specific pan‐caspase inhibitor Q‐VD‐oph decreased the percentage of propidium iodide‐positive neurons (52.5% ± 3.1%, 39.4% ± 3.5%, 44.6% ± 3%, respectively, vs. 65% ± 3% in glutamate + vehicle). EM studies showed mitochondrial release of cytochrome c in neurons in the early stages of necrosis and cleaved caspase‐3 immunoreactivity in morphologically necrotic neurons. These results suggest that an active mechanism contributes to the demise of a subpopulation of excitotoxic necrotic neurons.

FOCAL ENDOTHELIAL CELL DEGENERATION AND PROLIFERATIVE ENDARTERITIS IN TRAUMA-INDUCED EARLY LESIONS OF NECROBIOSIS LIPOIDICA DIABETICORUM

Microangiopalhy is an essential component in diabetic vascular pathology. We report ultrastructural observations of ballooning degeneration involving isolated endo-thelial cells of cutaneous capillaries, while leaving adjacent cndothclial cells relatively intact in six diabetic patients with early lesions of necrobiosis lipoidica induced by trauma. Focal proliferation of cndothclial cells encroaching upon the vascular lumina (obliterative endar-tcritis) was also observed. Lectin studies on biopsy specimens of older lesíons of neerobiosis lipoidica revealed paucity of dermal blood vessels. These observations enable us to gain further insight into the pathophysiological mechanisms that underlie diabetic microvascular disease.

Mitochondrial crowding in smooth muscle cells after arterial ligation

Although the ultrastructural appearance of mitochondrial crowding after ischemic injury has been reported in myocardial and skeletal muscles, the mechanism for this is not clear. Thirty-five branches of the mesenteric arteries removed from eight patients during surgery were examined by electron microscopy: 22 had ligatures applied 30 minutes to 4 hours before resection, 13 had no ligatures. The number of mitochondria per smooth muscle cell was significantly increased in the arterial segments under the ligature (ligated site) and immediately distal to the ligature (distal site) compared with the nonligated segments (control site). Mitochondria in ischemic cells were also noted to be swollen compared with those in nonischemic cells. Within the ligated site, paradoxically, the severity of ischemic injury correlated inversely with the increase in mitochondrial counts. In conclusion, mitochondria crowding after ischemic injury is due to both swelling and an increase in the number of organelles, and appears as early as 30 min after injury. The results also suggest that, in severely ischemic sites, cells capable of rapidly increasing their mitochondrial count may suffer less ischemic injury.

PREDOMINANCE OF CD8 SUBSET IN ID ERUPTION OF POISON OAK‐INDUCED DERMATITIS

The pathophysiology and immune mechanisms involved in the clinical syndrome of autoeczematization remain a mystery. In this study of nickel dermatitis without autoeczematization and poison oak dermatitis with autoeczematization, it was noted that the process of autoeczematization was associated with the presence of CD8+ lymphocytes within the epidermis and the expression of HLA‐DR antigens on epidermal keratinocytes. It is surmised that since CD8+ clones are induced by poison oak antigen but not by nickel, the inability of nickel to induce CD8+ lymphocytes may explain why uncomplicated nickel dermatitis does not autoeczematize. Since the selective adherence ofCD8+ lymphocytes to keratinocytes, probably via the expression of adhesion molecules such as ICAM‐1, the generation of antigens on endothelial cells of high endothelial venules involved in lymphocyte trafficking, and the expression of HLA‐DR antigens on epidermal keratinocytes are all due to the activity of interferon‐8, it is deduced that this Lymphokine may play a key role in id eruptions induced by contact allergens.

ACRODERMATITIS ASSOCIATED WITH ZINC DEFICIENCY: FEATURES AND POSTULATED MECHANISM

It is currently unexplained why zinc deficiency should result in a cutaneous eruption of such a distinctive and characteristic distribution (acrodermatitis), affecting the groin, periorificial surfaces and periungual areas of fingers and toes. In this study, we provide ultrastructural evidence suggestive of excessive damage of the epidermis by neutrophils, with other features resembling tissue damage from free radicals. We propose that these changes are consistent with a deficiency of superoxide dismutase, a zinc metalloenzyme, which is mainly responsible for neutralizing the destructive effects of the reactive oxygen species released by neutrophils. The various zymosans on many yeast cells, including Candida, activate complement, resulting in the generation of C5a, a potent chemattractant for neutrophils, and the location of the eruption in the characteristic sites in zinc deficiency may be explained by the natural occurrence of Candida albicans in these areas.