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On the organization of astrocytic gap junctions in rat brain as suggested by LM and EM immunohistochemistry of connexin43 expression.

Gap junctions and the intercellular communication syncytium they form between glial cells are thought to play a critical role in glial maintenance of appropriate metabolic environments in neural tissues. We have previously suggested (Yamamoto et al., Brain Res. 508:313-319, '90) that the vast majority of astrocytes in rat brain express connexin43, one of several recently identified gap junction proteins. Here, we confirm ultrastructurally that astrocytes in a number of brain regions of rat are immunolabelled with an antibody against connexin43 and that neurons and oligodendrocytes are devoid of labelling. The distribution of connexin43 immunoreactivity throughout the brain is presented at the light microscope (LM) level. By LM, immunoreactive structures consisted primarily of round or elongated puncta ranging from 0.3 microns to 4 microns in length and of annular profiles ranging from 1 to 10 microns in diameter. Immunolabelled fibrous processes were only occasionally seen and no labelling was observed in astrocytic cell bodies. Long, linear arrays of puncta were rare in gray matter but were common in white matter where they were arranged parallel to myelinated fibers. Puncta organized in a honeycomb pattern were seen near the cerebral cortical surface and frequently around blood vessels. Regional immunoreaction density, which was a reflection of either the concentration or staining intensity of immunoreactive elements, was remarkably heterogeneous; dramatic differences in labelling intensity frequently delineated anatomical boundaries between adjacent nuclei. Abrupt as well as graded fluctuations of immunoreaction intensity were also observed within nuclear structures. By electron microscopy (EM), gap junctions of fibrous and protoplasmic astrocytes were intensely stained and labelled organelles were often observed intracellularly in areas near gap junctions. These junctions and the spread of immunoreaction product to perijunctional organelles in their vicinity were considered to correspond to puncta seen by LM. Labelling within astrocytic cell bodies was seen in only a few instances. In some brain areas, astrocytic processes commonly gave rise to immunoreactive lamellae that partially ensheathed neuronal cell bodies, axon terminals, dendrites, and synaptic glomeruli. Such lamellae were considered to correspond to immunoreactive annular profiles seen by LM. Perivascular endfoot processes of astrocytes displayed intense staining of their gap junctions and portions of their apposing membranes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Neuroglial arrangements in the olfactory glomeruli of the hedgehog.

The olfactory glomeruli represent morphological and functional units in which olfactory information is processed in specialized synaptic arrangements established between the central processes of sensory neurons, whose cell bodies are located in the olfactory epithelium, and the terminal (intraglomerular) portions of the dendrites of periglomerular, tufted, and mitral cells. The olfactory glomeruli are surrounded by distinctive glial formations in which the peripheral glia interacts with the central glia. We have studied the morphology and organization of neuroglial cells in the layer of olfactory nerves and the glomerular layer of the olfactory bulb in the insectivorous hedgehog (Erinaceus europaeus) with the electron microscope, Golgi method, and immunohistochemistry by using antibodies to glial fibrillary acidic protein (GFAP) and "rip," a monoclonal antibody that stains oligodendrocytes and their processes in the rat (Friedman et al.: Glia 2:380-390, '89). The peripheral glia is represented by a special category of cells that are closely related to astrocytes and known as sheathing cells. They accompany olfactory axons to their entrance in the glomeruli where they interact with the central glia, represented by astrocytes and oligodendrocytes. The sheathing cells typically display indented nuclei and protoplasmic expansions forming laminar processes wrapping several axons together. Astrocytes surrounding the glomerular neuropil belong to the velate type. They display numerous sheet-like processes enveloping dendritic segments and periglomerular cell bodies. Oligodendrocytes were found surrounding the glomeruli and at the interstices separating different glomeruli. Myelinated dendritic segments and cell bodies were found surrounding the olfactory glomeruli. These myelin coverings probably derive from oligodendrocytes. Together with the astrocytic lamellar expansions, they provide a rigid structural support that contributes to the segregation of group of different cells while remaining relatively isolated from other influences at the periphery of the glomeruli.

Animals↗

Postnatal development of vimentin-positive cells in the rabbit superior colliculus.

The present study examined the postnatal development of the radial glia in the rabbit superior colliculus during the first 40 postnatal days. An antivimentin monoclonal antibody and the carbocyanine fluorescent tracer DiI were used in order to investigate the development of laminar connectivity in the superior colliculus. We focused our study on the superficial gray layer, the intermediate layer, and the deep layers of the superior colliculus, the periaqueductal gray matter (PAGM), and the medial intercollicular region. Vimentin-positive structures of glial lineage consisted of 1) the main radial system, which in the newborn rabbit was made up of wavy fibers that ran from the aqueduct to the pial surface, where they terminated in end-feet. At postnatal day 15, these fibers diminished to 100-200 microns long wavy tracts, which emanated from the aqueduct, and to a few straight or arched fragments in the superficial gray layer; 2) the median ventricular formation, which extends from mesencephalic aqueduct to the intercollicular sulcus, was characterized by a series of ascending, vimentin-positive fibers, some of large caliber, which persisted until postnatal day 40; 3) the tangential fiber system, which was made up of fibers that diverged from the median ventricular formation and of a number of short tracts running perpendicular to the periaqueductal radial fibers; these structures may provide support for migrating subpopulations of neurons; 4) immature and mature-like protoplasmic and fibrous astrocytes, which appeared during the second postnatal week. Thereafter, the number of vimentin-positive astrocytes decreased sharply. Our findings generally support earlier descriptions of the radial glia, except for the persistence, in superficial layers of the superior colliculus, of straight and curved fragments of fibers, which may participate in the organization of visual afferents at this level.

Aging↗

Correction of antigen-specific T cell defects in aged murine gut-associated lymphoid tissues an immune intervention by combined adoptive transfer of an antigen-specific immunoregulatory CD4 T cell subset and interleukin 2 administration.

Gut-associated lymphoid tissues (GALT) from aged mice enterically immunized with Mycobacterium paratuberculosis protoplasmic antigen show hyperreactive humoral immune responses; this hyperresponsiveness can be corrected to a considerable extent, but not entirely, by systemic administration of interleukin 2 (IL 2) alone. The aim of the present study was to determine further whether the hyperreactivity in the antigen-specific humoral immune responses in aged GALT could be fully restored by adoptive transfer of in vitro expanded antigen-specific IL 2-dependent helper (CD4+VV-) T cells from GALT in conjunction with recombinant IL 2 administration. The results show that the age-associated hyperresponsiveness in gut mucosal antigen-specific humoral immune responses can be entirely corrected by adoptive transfer with the antigen-specific GALT T helper cells together with in vivo IL 2 administration. The mechanism of this restoration involves reversal of the decline in antigen-specific CD8+ suppressor T cell functions in aged GALT.

Aging↗

Expression of beta-adrenergic receptors by astrocytes isolated from adult rat cortex.

Astrocytes are the most numerous cell type in the central nervous system. When cultured, these cells express a wide variety of receptors for neurotransmitters. Nonetheless, it has yet to be firmly established that adult astrocytes in situ express receptors for neurotransmitters. In this report the technique of combined receptor autoradiography and immunocytochemistry has been applied to isolated astrocytes from adult animals to examine beta-adrenergic receptor (beta-AR) expression by these cells. It is shown that fibrous and protoplasmic astrocytes isolated from adult rat cerebral cortices specifically bind the beta-AR ligand iodocyanopindolol and that this binding is inhibited by the beta-AR ligands propranolol and isoproterenol. These results indicate that at least two of the major morphological subtypes of adult astrocytes express beta-ARs.

Animals↗

An endogenous lectin found in rat astrocyte cultures has a role in cell adhesion but not in cell proliferation.

The presence of an endogenous cerebellar soluble lectin (CSL) has been demonstrated in cultured rat astrocytes by using immunocytochemical techniques. In these cells, the location of lectin CSL was found intracellularly as well as on the external surface of the plasma membrane of the cell bodies and processes, especially in the zones of contact between cells. This suggested that CSL could have a role in adhesion of astrocytes to sister cells. Kinetics of adhesion of astrocytes to culture dishes precoated with CSL showed a rapid binding of these cells. In confluent astrocyte cultures, anti-CSL Fab fragments affected the shape and organization of astrocytes (retraction of the cytoplasm), but they did not detach cells from the substratum. These results indicated that CSL has adhesive properties for astroglial cells and is probably involved 1) in adhesion of astrocytes to sister cells; 2) in binding of protoplasmic regions of astrocyte membrane to the substratum. Further support for these roles came from demonstration of the presence in cultures of glycoprotein ligands recognized by this lectin. The problem of the mitogenic properties of the lectin was also questioned. The addition of CSL to confluent astroglial cultures was able to stimulate only by 40% the proliferation of these cells at an optimal concentration of 5 micrograms CSL lectin/ml of culture medium. This indicated that CSL is not a powerful growth factor for astrocytes.

Animals↗

Comparative immunohistochemical study of glial filament proteins (glial fibrillary acidic protein and vimentin) in goldfish, octopus, and snail.

Glial fibrillary acidic protein (GFAP) and vimentin proteins are known to be component proteins of glial filaments in the CNS of many vertebrates. The nature of the filaments present in the glial cells of the goldfish optic tectum and in the CNS of two members of the Mollusca (Helix pomatia and Octopus vulgaris) were investigated using immunocytochemical localization of monoclonal antibodies to GFAP and vimentin. Immunoblots visualized by the alkaline phosphatase method showed cross-reactive protein bands to GFAP and vimentin antibodies in total brain homogenates of the goldfish, octopus, and snail. Immunofluorescence staining of the goldfish optic tectum showed GFAP immunoreactivity, primarily in the ependymal cell processes. Immunogold labelling at the ultrastructural level verified that GFAP antibodies were bound to glial filaments. Immunolabelling of the optic lobe of Octopus vulgaris and the cerebral ganglia of Helix pomatia suggests that a protein exhibiting antigenic properties similar to GFAP is a component protein in the filaments of the protoplasmic and filamentous glia randomly distributed throughout the CNS. Unlike anti-GFAP antibodies, which stained relatively specific to filaments, vimentin staining in the CNS tissues of the three organisms studied did not appear to be exclusive to filamentous structures. As vimentin protein has been shown, in previous studies as well as our own, to exist in many tissue types, this suggests that it does not appear to be confined to glial filaments but is shared with other subcellular components. The proteins GFAP and vimentin which are thought to be well conserved in vertebrate evolution also appear to be expressed in the nervous system of some lower organisms.

Animals↗

Comparative biochemical, morphological, and immunocytochemical studies between C-6 glial cells of early and late passages and advanced passages of glial cells derived from aged mouse cerebral hemispheres.

We have used C6 glial cells (2B clone), early and late passage, as well as advanced passages (8-17) of glial cells derived from aged (18-month-old) mouse cerebral hemispheres (MACH), as model systems for studying glial properties. In this study passages 20-24 were considered "early" and passages 73-90 were considered "late." Activities of glutamine synthetase (GS) and cyclic nucleotide phosphohydrolase (CNP) were used as biochemical markers for astrocytes and oligodendrocytes, respectively. Glial phenotypes were identified immunocytochemically using double staining for glial fibrillary acidic protein (GFAP) and A2B5 antigen (type 1 and type 2 astrocytes) or galactocerebroside (GalC) and A2B5 antigen (oligodendrocytes); cells positive for A2B5 and negative for both GFAP and GalC were considered to be precursor cells. Cultures were grown either in DMEM supplemented with 10% fetal bovine serum or in serum-free chemically defined medium (CDM) supplemented with insulin and transferrin. We report that early-passage C6 glial cells continue to be bipotential cells and when grown in the absence of serum express high GS and CNP activities correlating with the high number of GFAP- and GalC-positive cells, respectively. Late-passage cells continued to be committed to the type 2 astrocytic phenotype regardless of media composition (+/- serum). MACH cultures consist of protoplasmic type 1 astrocytes, differentiated type 2 astrocytes, and oligodendrocytes as well as glial progenitor cells. When these cultures were grown in CDM+transferrin, both GS and CNP activities increased, suggesting that transferrin has provided the signal for progenitor cells present in these cultures derived from aged brain to differentiate into type 2 astrocytes and oligodendrocytes.

Aging↗

Epstein-Barr virus-related ultrastructural modifications of plasma membrane during B-cell transformation.

Ultrastructural modifications are described in the plasma membrane of in vitro established human B cells. By the freeze-fracture technique, intramembrane particles (IMPs) are quantified in B lymphocytes following Epstein-Barr virus (EBV) transformation in vitro, and in B-lymphoma (Burkitt-type) cells, either positive or negative for EBV genome. Analysis shows an overall increase in IMP density as compared to normal controls. Differences are observed between the protoplasmic and exoplasmic faces of fractured membranes as well as among in vitro transformed and clearly neoplastic cells. Results indicate that conformational changes in IMP distribution parallel neoplastic evolution of transformed cells.

B-Lymphocytes↗

Simultaneous demonstration of glia- and glioma-associated antigens in human astrocytomas.

Glial fibrillary acidic protein (GFAP) and glioma-associated antigens (GAA) defined by monoclonal antibodies (MAbs) were demonstrated simultaneously in human astrocytoma tissue. GFAP was stained by PAP-method, GAA were visualized by avidin-biotin-technique using alkaline phosphatase. In primary and secondary tumors as well as in tissue culture heterogeneity of GFAP- and GAA-expression is obvious. GFAP is mostly restricted to cell processes and less marked in the perinuclear space. Depending on the individual antibody, MAbs-positive material is located either in the tumor cell plasma (MUC 8-22) or on cell surface membranes (MUC 2-63). There is remarkable expression of GAA in cell clusters which fail to express GFAP. At higher magnification, 3 types of cellular reactivity are detectable: (a) cells which react only with anti-GFAP, (b) cells which react only with anti-GAA and (c) cells which express both, GFAP and GAA, especially those of protoplasmic astrocyte type. These cells also occur in subcutaneous tumor grafts, and may thus represent not only a reactive event, but be part of tumor cell populations.

Animals↗

Visualization of the inner and outer surfaces of the cell membrane and cytoskeleton by polyethylene glycol embedding, subsequent deembedding, and rotary replication with platinum.

Using rotary replication with platinum and carbon to embedment-free sections of polyethylene glycol (PEG)-embedded tissue from which PEG had been removed, the membrane specializations on the outer (exoplasmic) and inner (protoplasmic) cell surface as well as the organization of the cytoskeleton was demonstrated. The high quality and excellent preservation were comparable to that obtained by the rapid-freezing, deep-etched replica method. The present results indicate that the PEG method is essentially acceptable as a reliable morphological technique. Because either sectioned or replica images from the same tissue cells can be observed simultaneously, the PEG method with rotary replication should provide valuable information on cell ultrastructure.

Animals↗

Immunogold localization of actin in the testis and exocrine pancreas: spatial relationship with tight junctional strands.

The fracture-label technique was used in conjunction with a monoclonal antibody to actin and the phospholipase A2-colloidal gold (PLA2-CG) method to examine the spatial distribution of actin filaments in relation to the three-dimensional arrangement of tight junctional strands in rat testes and exocrine pancreatic acinar cells. The intimate association of actin filaments with tight junctional strands in the pancreas and testis was also illustrated by a double-labeling experiment in which freeze-fractured pancreas or testis was labeled with monoclonal antibody-protein A-gold (30 nm gold size) followed by incubation with a PLA2-CG complex (11 nm gold size). Freeze-fracture-exposed tight junctional strands in both testicular and exocrine pancreatic cells labeled by PLA2-CG complex, indicated the presence of phospholipids in these cylindrical membranous structures. Immunolabeling of freeze-fractured testes with a monoclonal antibody to actin revealed a narrow band of gold particles juxtaposed to the cytoplasmic aspect of the protoplasmic membrane halves decorated with parallel linear arrays of cylindrical tight junctional strands. Many of the gold particles representing actin antigenic sites were in direct contact with the cross-fractured tight junctional strands. Fracture-label preparations of exocrine pancreas labeled with the monoclonal anti-actin antibody also exhibited a similar labeling pattern at the apex of acinars cells where the tight junction complex is located. Double-labeling experiments revealed the simultaneous labeling of actin and phospholipids in the same fracture-label preparations. Digestion of testicular and pancreatic tissue samples in a free PLA2 solution prior to labeling with the monoclonal antibody or PLA2-CG complex removed not only the gold labeling previously seen over the tight junctional strands but also reduced drastically the immunolabeling for actin that was previously seen associated with the tight junction complex. Taken together, results of the present study showed that actin filaments are structural components of the tight junction strands and are connected to the cytoplasmic aspect of the latter structures. The interaction between this particular cytoskeletal element and the tight junction may be through the binding of a special domain of the actin filament to the phospholipids that partially make up the tight junctional complex.

Actins↗

Dynamic study of intramembranous particles in human fresh erythrocytes using an "in vitro cryotechnique".

For analyses of dynamic ultrastructures of erythrocyte intramembranous particles (IMPs) in situ, a quick-freezing method was used to stabilize the flow behavior of erythrocytes embedded in vitreous ice. Fresh human blood was jetted at various pressures through artificial tubes, in which the flowing erythrocytes were elongated from biconcave discoid shapes to elliptical ones, and quickly frozen in liquid isopentane-propane cryogen (-193 degrees C). They were freeze-fractured using a scalpel in liquid nitrogen, and routinely prepared for replica membranes. Many IMPs were observed on the protoplasmic freeze-fracture face (P-face) of the erythrocyte membranes. Some control erythrocytes under nonflowing or stationary conditions showed IMPs with their random distribution. However, other jetted erythrocytes under flowing conditions showed variously sized IMPs with much closer distribution. They were also arranged into parallel rows in some parts, and aggregated together. This quick-freezing method enabled for the first time the visualization of time-dependent topology and the molecular alteration of IMPs in dynamically flowing erythrocytes.

Cryoelectron Microscopy↗

Quantitative analysis of intramembranous particles in the membranous system of rat liver cells at different stages of development and aging.

The density of intramembranous protein particles was studied by freeze-fracture. Particle density on the fracture faces of the plasmalemma and the rough endoplasmic reticulum (RER), as well as the outer and inner membranes of the nucleus and the mitochondria in rat hepatocytes were quantified. Comparison among different age groups sampled days postcoitum (dpc), days postpartum (dpp), and months postpartum (mpp) showed age-related changes in particle density in each membrane system. With the exception of the RER, particle densities increased after the 16th dpc, reached a maximum at birth, and then decreased with increasing age. Simultaneously, the number of nuclear pores shows a positive correlation with the particle density of the nuclear membranes. The particle density on the membranes of the RER shows a maximum on the 16th dpc and on the 6th dpp. Thereafter, the density of the RER decreases slightly. In all membrane systems, the density of the particles on the external fracture faces is more variable than density of the particles on the protoplasmic fracture faces.

Aging↗

Synaptic organization of dorsal area of the turtle, Pseudemys scripta elegans.

The dorsal ventricular ridge is a subcortical structure receiving sensory information from the thalamus in reptiles. In the red-eared turtle, Pseudemys scripta elegans, it contains four cytoarchitectonic areas each characterized by distinct thalamic projections. This is an electron microscopic study of one of these, the dorsal area, which receives its thalamic input from the tectorecipient nucleus rotundus. It contains four concentric zones, internal to the ependymal zone, each of which is distinguished by the distribution of spiny and aspiny neurons. The ependymal zone of dorsal area contains tanycytes whose tails extend into zones 2 and 4. Synapses, usually with asymmetric junctional complexes and round synaptic vesicles, occur on these processes. Zone 1 neurons have fusiform somata and dendrites that parallel the ventricular surface. Their cytoplasm contains rough endoplasmic reticulum located primarily in Nissl bodies, lipofuchsin granules, multivesicular bodies, extensive arrays of Golgi apparatus, and large numbers of mitochondria. Synapses occur mainly on dendritic spines and shafts of zone 1 neurons and less frequently on somata. The majority have round vesicles and asymmetric junctional complexes. In contrast to those in zone 1, neurons in zones 2 and 4 have large amounts of rough endoplasmic reticulum, giving their cytoplasm an electron-dense quality. Synapses occur mainly on spines and shafts of zone 2 and 4 neurons. As in zone 1, the majority have round synaptic vesicles and contain asymmetric junctional complexes. Zones 2 and 4 contain clusters of neurons distributed among isolated neurons. The clusters are larger and less frequent in zone 2. Protoplasmic and fibrous glial processes, axon boutons, dendrites, and axon fascicles surround the neuron clusters. Though less numerous, the same structures also occur inside the clusters. Most synapses inside the clusters have round synaptic vesicles, asymmetric junctional complexes, and occur mainly on spines. Some neurons in clusters have somata whose plasma membranes are in direct apposition. In contrast to dorsal ventricular ridge in snakes, no specialized intercellular contacts were seen between somata in clusters.

Animals↗

Expression of inducible nitric oxide synthase and cyclooxygenase-2 after excitotoxic damage to the immature rat brain.

It is well established that after adult brain damage the enzymes cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) play an important role in the inflammatory processes and oxidative stress, which are considered to be the leading factors contributing to delayed cell death. The contribution of these enzymes to postnatal brain damage, however, is poorly understood. In our study, excitotoxic lesions were induced by the injection of N-methyl-D-aspartate in the cortex of postnatal day 9 rats. After different survival times ranging from 4 hr to 7 days post-lesion, brain sections were processed for the immunocytochemical demonstration of COX-2 and iNOS and double labeling with neuronal, glial and neutrophil markers. First and maximal de novo induction of iNOS and COX-2 expression was found at 10 hr post-lesion. Expression of both enzymes started to diminish at 24 hr, reaching basal levels at day 3. iNOS-expressing cells were mainly identified as infiltrated neutrophils as well as highly ramified protoplasmic astrocytes closely associated with blood vessels. Moreover, scattered iNOS-positive neurons were found at the lesion borders. In contrast, COX-2 was mainly observed in reactive microglial cells and neuronal cells. COX-2-positive neurons were found within the degenerating area at 10 hr and at the borders of the lesion later on. This study shows that maximal iNOS and COX-2 expression precedes the period of massive neuronal death observed at 24 hr post-lesion, and may therefore contribute to the evolution of the inflammatory response and the neurodegenerative process after an excitotoxic lesion to the postnatal brain.

Animals↗

Rapid up-regulation of the neuronal serotoninergic phenotype by brain-derived neurotrophic factor and cyclic adenosine monophosphate: relations with raphe astrocytes.

Up-regulation of the neuronal serotoninergic phenotype in relation to astrocytic population was studied in primary cultures of rat embryonic rostral raphe. Short treatments (18 hr at day in vitro 4) with brain-derived neurotrophic factor (BDNF) or dibutyryl-cAMP (dBcAMP) increased the number of serotoninergic neurons by approximately 80% and approximately 40%, respectively, and markedly enhanced the branching (by 11-fold and 5-fold, respectively) and total length (by 4-fold and 2.5-fold, respectively) of their neurites. Concomitantly, under BDNF treatment, the astrocyte population was decreased by half and became mostly protoplasmic-like. In contrast, dBcAMP treatment also reduced the astrocytic cell density (by one-third) but induced a stellate morphology. Similar short treatment with the astrocyte-derived S100beta factor induced no modification of the serotonin (5-HT) neuronal phenotype nor of astrocytes morphology. Both BDNF- and cAMP-induced effects were abolished by simultaneous treatment with the specific tyrosine kinase inhibitor genistein, suggesting a role for the high-affinity BDNF receptor tyrosine kinase (TrkB). These data suggest that BDNF and cAMP, but not S100beta, rapidly induce both an up-regulation of the 5-HT neuronal phenotype and modifications of the neighboring astrocytes in a TrkB-dependent manner.

Animals↗

Histochemical localization of carbonic anhydrase in vertebrate nervous tissue.

The histochemical method of Häusler was employed to demonstrate carbonic anhydrase (CA, carbonate hydrolase, 4.2.1.1.) in tissue sections. The CA reaction was inhibited in the presence of 5 mM acetazolamide. In the frog and fish retinas the CA activity was positive in the Müller fiber, the laminated segment of the cones and the myelin of the axons, and was negative in the laminated segment of the rods, the photoreceptor ellipsoid and myoid, the horizontal, bipolar, amacrine and ganglion cells, and the pigment epithelium. In the spinal cord of cat and fish the CA activity was positive in the myelin of the axons, the perineuronal oligodendroglia and the protoplasmic astrocytes, and was negative in the cell body, dendrite, and axon of the neurons and in the fibrous astrocytes. In the dorsal root ganglion of the cat CA reaction was positive in the satellite cells and the myelin, and was negative in the neuronal cell body and its processes. The capillaries in central nervous tissue show no CA reaction. The erythrocyte is CA positive while the mitochondria are CA negative.

Acetazolamide↗