Acid Phosphatase Enzyme Histochemistry Research Articles

MTOR-dependent abnormalities in autophagy characterize human malformations of cortical development: evidence from focal cortical dysplasia and tuberous sclerosis

Focal cortical dysplasia (FCD) is a localized malformation of cortical development and is the commonest cause of severe childhood epilepsy in surgical practice. Children with FCD are severely disabled by their epilepsy, presenting with frequent seizures early in life. The commonest form of FCD in children is characterized by the presence of an abnormal population of cells, known as balloon cells. Similar pathological changes are seen in the cortical malformations that characterize patients with tuberous sclerosis complex (TSC). However, the cellular and molecular mechanisms that underlie the malformations of FCD and TSC are not well understood. We provide evidence for a defect in autophagy in FCD and TSC. We have found that balloon cells contain vacuoles that include components of the autophagy pathway. Specifically, we show that balloon cells contain prominent lysosomes by electron microscopy, immunohistochemistry for LAMP1 and LAMP2, LysoTracker labelling and enzyme histochemistry for acid phosphatase. Furthermore, we found that balloon cells contain components of the ATG pathway and that there is cytoplasmic accumulation of the regulator of autophagy, DOR. Most importantly we found that there is abnormal accumulation of the autophagy cargo protein, p62. We show that this defect in autophagy can be, in part, reversed in vitro by inhibition of the mammalian target of rapamycin (mTOR) suggesting that abnormal activation of mTOR may contribute directly to a defect in autophagy in FCD and TSC.

Comparative nephrotoxicity of cisplatin and new octahedral Pt(IV) complexes

Previously, we have reported that the newly synthesized octahedral Pt(IV) compound, trans,cis-Pt(acetato)(2)Cl(2)(1,4-butanediamine), K101 and trans,cis-Pt(trifluoroacetato)(2)Cl(2)(1,4-butanediamine), K102 showed potent antitumor activities in vitro and in vivo. In order to compare the nephrotoxicity of the newly synthesized Pt(IV) complexes, K102 and K102 with cisplatin, we performed various tests. We performed a single dose acute toxicity test for LD(50) values determination, biochemical assays in blood serum, acid phosphatase enzyme histochemistry and transmission electron microscopic studies in renal proximal tubular cells in mice in vivo. The route of drugs administration is intraperitoneal injection. In biochemical assays, the serum levels of BUN were significantly elevated at 6 h (p < 0.001), 1 day (p < 0.05) and 3 days (p < 0.001) after injection in cisplatin treated mice (6 mg/kg, single dose, i.p.). On the other hand, the serum levels of BUN were slightly elevated at 6 h (p < 0.01) only in K101 treated mice (8.2 mg/kg, single dose, i.p.), and were significantly raised at 6 h, 1 and 3 days (p < 0.05) after injection in K102 treated mice (6.2 mg/kg, single dose, i.p.). The higher serum BUN level in K102 treated mice is considered that K102 possesses more lipophilic fluoro group than acetyl group in K101. The values of creatinine and uric acid were similar in all groups. The ultrastructural morphological changes of K101- or K102-administrated mice were less remarkable than cisplatin-administrated mice. In acid phosphatase enzyme histochemistry, cisplatin treatment induced relevant changes in the distribution pattern of enzyme activity compared with K101 or K102 treatment at 7 days after injection. In conclusion, these results show that K101 is less nephrotoxic than cisplatin and a promising new platinum complex.

Disruption of Erythrocytes Distinguishes Fixed Cells/Tissues from Viable Cells/Tissues Following Microwave Coagulation Therapy

Microwave coagulation therapy (MCT) has recently been applied to treat hepatic tumors. However, the histological changes in the liver following MCT have not been fully elucidated. A type of cell death known as microwave fixation has been reported in areas adjacent to the microwave irradiator electrodes, and these areas are without acid phosphatase (AcP) activity. Diagnosis of microwave-fixed tissue by hematoxylin and eosin (HE) staining is very difficult because morphology is well maintained for months. In an effort to clarify the histological changes and the mechanisms of microwave fixation, we performed HE staining, enzyme histochemistry for AcP, and electron microscopy in both rat and human liver samples after MCT. Although the microwave-fixed tissues maintained their structure on HE staining, membranes of microwave-fixed cells were seriously damaged and there were no apparent organelle structures in these cells on electron microscopy. Erythrocytes were also damaged in these tissues on both light and electron microscopy. The cause of microwave fixation is thought to be injury of the membrane, which is similar to coagulative necrosis. In conclusion, microwave fixation can be considered a type of coagulative necrosis without enzyme digestion. Disruption of erythrocytes on HE staining is an interesting and important diagnostic clue in distinguishing nonviable fixed tissues from viable tissues following MCT.

Effects of microwave irradiation on rat hepatic tissue evaluated by enzyme histochemistry for acid phosphatase.

Microwave coagulation therapy (MCT) has been applied to small hepatic carcinomas. To clarify the sequential changes in histology and the viability of the microwave-irradiated tissue, we examined irradiated normal rat liver with enzyme histochemistry for acid phosphatase (AcP). In the samples immediately after irradiation, the margin of the irradiated region was indistinct in H&E stain, while AcP enzyme histochemistry disclosed well-demarcated distinct zones: an inner zone adjacent to the electrode without AcP activity and a surrounding outer zone with attenuated enzyme activity. In the inner zone, the nuclear staining with hematoxylin persisted for at least one month, whereas that in the outer zone disappeared 24 hr after irradiation and was accompanied by neutrophilic infiltration and then replaced by granulation tissue. Our results indicated that microwave irradiation caused tissue fixation in the inner zone and coagulative necrosis in the outer zone. Because microwave-fixed cells retained their morphology well, they appeared very similar to normal cells in H&E-stained section. Enzyme histochemistry may be useful for assessment of cellular viability after microwave irradiation, by enabling us to distinguish fixed cells from viable cells.

SSeCKS immunolabeling in rat primary sensory neurons

SSeCKS (src suppressed C kinase substrate) is a protein kinase C substrate that may play a role in tumor suppression. Recently described in fibroblasts, testes and mesangial cells, SSeCKS may have a function in the control of cell signaling and cytoskeletal arrangement. To investigate the distribution of SSeCKS throughout the nervous system, representative sections of brain, spinal cord and dorsal root ganglia were processed using immunofluorescence. Labeling of central axonal collaterals of primary sensory neurons was observed in the dorsal horn at all spinal levels. SSeCKS-immunoreactivity was also observed in the cerebellum, medulla and sensory ganglia (including trigeminal ganglia). The pattern and distribution of anti-SSeCKS labeling in dorsal root ganglia and the dorsal horn of the spinal cord was similar to that observed for other markers of small primary sensory neurons. Therefore, the coexistence of SSeCKS with substance P, CGRP and acid phosphatase was examined in sections of sensory ganglia, spinal cord and medulla using double immunofluorescent labeling for SSeCKS and substance P/CGRP or sequential SSeCKS immunofluorescence and acid phosphatase/fluoride-resistant acid phosphatase enzyme histochemistry. A small portion of the SSeCKS-labeled cell bodies appeared to represent a subpopulation of substance P (4.8%) and CGRP (4.7%) containing neurons, while 45.0% contained fluoride-resistant acid phosphatase reactivity. These results indicate that SSeCKS has a restricted distribution within the nervous system and that expression of this protein may reflect the specific signaling requirements of a distinct population of nociceptive sensory neurons.

Enzyme histochemistry can assese the effects of microwave irradiation on rat hepatic tissue

Tabuse reported that the microwave ablation elicited thermal damage of tissue that resulted in coagulation necrosis. While some researchers pointed out the possibility that some tumor cells remain viable in the ablated area. To clarify the sequential histologic of the microwave-irradiated tissue, we examined irradiated normal rat liver with enzyme histochemistry for acid phosphatase. The irradiated region was composed of histologically different two distinct areas : a central area adjacent to the electrode and a peripheral area. Cellular morphology and time sequence of tissue reaction in the peripheral area were consistent with coagulation necrosis. On the other hand, in the central area microwave caused tissue fixation. Because microwave fixed cells which were similar to normal cells in HE stain, enzyme histochemistry that enables us to distinguish fixed cells from viable cells may be useful to assess the effects of microwave irradiation.

Differentiation of necrotic cell death with or without lysosomal activation: application of acute liver injury models induced by carbon tetrachloride (CCL4) and dimethylnitrosamine (DMN).

We investigated the relationship between DNA degradation and lysosome activity (loss of lysosomal integrity) in necrotic cell death induced by carbon tetrachloride (CCl4) and dimethylnitrosamine (DMN): coagulation necrosis and hemorrhagic necrosis, respectively. TdT-mediated dUTP-biotin nick end-labeling (TUNEL) and enzyme histochemistry for acid phosphatase were performed in both models and results were analyzed by light microscopy, electron microscopy, and confocal laser scanning microscopy (CLSM). In the CCl(4)-injected liver, TUNEL staining was closely associated with release of lysosomal enzymes into the cytoplasm, and intranuclear deposition of lysosomal enzymes took place at an early stage of subcellular damage. In the DMN-injected liver, TUNEL-positive nuclei tended to have well-preserved lysosomes and centrally localized TUNEL signals. It was assumed that acute hepatocellular damage in the CCl4-injected liver would be characterized by necrotic cell death with lysosome activation and that damage in the DMN-injected liver would be necrotic cell death without lysosome activation. In the DMN-injected liver, DNA degradation may be selectively induced in the nuclear center, in which heterochromatin (including inactive chromatin) is believed to be a target. We concluded that necrotic cell death, i.e., DNA degradation, would be at least divided into two types, with/without association with lysosome activation, represented by necrotic cell death in the CCl4-injected liver and that in the DMN-injected liver.

Involvement of apoptosis and lysosomal hydrolase activity in the oviducal regression during induced molting in chickens: a cytochemical study for end labeling of fragmented DNA and acid phosphatase

Induced molting improves egg producing functions in hens. We investigated the mechanism of oviducal regression during induced molting. Involvement of apoptosis and autolysis in the oviducal regression process was analyzed by terminal deoxynucleotidyl transferase (T'dt)-mediated biotinylated deoxyuridine triphosphates (dUTP) nick end-labeling TUNEL) and an enzyme histochemistry for acid phosphatase. Nuclei positive for TUNEL were negligible and acid phosphatase staining was weak in the oviduct of laying hens. The frequency of TUNEL-positive nuclei was significantly increased in tubular gland cells of magnum, isthmus, and shell gland 2 d after cessation of egg laying and significantly decreased thereafter. The intensity of acid phosphatase staining was gradually increased during oviducal regression and extremely high on Day 7 after cessation of egg laying. These results suggest that during oviducal regression in induced molting hens, apoptosis is induced in the earlier stage of oviducal regression and autolysis occurs thereafter eventually, the glandular cells disappear.

Correlation between GAP43 and nerve growth factor receptors in rat sensory neurons

In mature rat sensory neurons, expression of the gene for the growth-associated protein, GAP43, was studied by in situ hybridization with a cDNA probe. Among neurons in normal lumbar dorsal root ganglia, labeling for GAP43 mRNA was heterogeneous, approximately one-half of the neurons being densely labeled. To characterize the latter population, individual neurons were examined in adjacent sections processed either for GAP43 hybridization or NGF-receptor radioautography. Virtually all neurons with high-affinity NGF binding sites had high basal levels of GAP43 mRNA and most GAP43-positive neurons bore NGF receptors. Another NGF-responsive population, sympathetic neurons in the superior cervical ganglion, also had high basal concentrations of GAP43 mRNA. Further co-localization studies in dorsal root ganglia were performed with immunohistochemistry for somatostatin and enzyme histochemistry for acid phosphatase. The latter 2 groups of sensory neurons have been previously shown to lack high-affinity receptors and were here shown to have low basal concentrations of GAP43 mRNA. From this and earlier studies, it can be assumed that substance P-immunoreactive neurons and strongly positive CGRP neurons synthesize GAP43 at high basal rate. One week following peripheral nerve transection, almost all neurons had high concentrations of GAP43 mRNA without correlation with NGF binding. Intrathecal infusion of NGF after the sciatic nerve was cut did not strongly influence this post-traumatic elevation in GAP mRNA. In normal dorsal root ganglia, neurons that have high-affinity NGF binding sites and are therefore potentially responsive to NGF also have high basal rates of synthesis of GAP43.(ABSTRACT TRUNCATED AT 250 WORDS)

Combined immunological and histochemical analysis of skin and lymph node lesions in histiocytosis X.

The immunological phenotype of the cells involved in skin and lymph node lesions from two cases of histiocytosis X (H-X) were analysed by immunofluorescence techniques using combinations of heterologous and monoclonal antisera to Ia-like antigen and human cortical thymocyte (HTA-1) determinant. These cells were also characterised by a new technique using simultaneous immunofluorescence and enzyme histochemistry for acid phosphatase (ACPase). The major cell type in the lesions was found to express the same Ia+, HTA-1+ phenotype as normal epidermal Langerhans' cells (LC) and was unreactive for ACPase. Additional cell types included Ia-, HTA-1- multinucleate giant cells and residual lymphoid populations. These findings endorse previous concepts that H-X is a proliferation of abnormal LC and emphasise the heterogeneous nature of the cells involved in the disease.