Tyrosine kinases and osmolyte fluxes during hyposmotic swelling

Recent evidence documents the involvement of protein tyrosine kinases (TK) in the signalling network activated by hyposmotic swelling and regulatory volume decrease. Both receptor type and cytosolic TK participate as signalling elements in the variety of cell adaptive responses to volume changes, which include adhesion reactions, reorganization of the cytoskeleton, temporal deformation/remodelling of the membrane and stress-detecting mechanisms. The present review refers to the influence of TK on the activation/operation of the osmolyte efflux pathways, ultimately leading to cell volume recovery, i.e. the osmosensitive Cl- channel (Cl-swell), the K+ channels activated by swelling in the different cell types and the taurine efflux pathway as representative of the organic osmolyte pathway.     

Brain volume regulation in response to changes in osmolality

Hypoosmolality and hyperosmolality are relatively common clinical problems. Many different factors contribute to the substantial morbidity and mortality known to occur during states of altered osmotic homeostasis. The brain is particularly vulnerable to disturbances of body fluid osmolality. The most serious complications are associated with pathological changes in brain volume: brain edema during hypoosmolar states and brain dehydration during hyperosmolar states. Studies in animals have elucidated many of the mechanisms involved with brain adaptation to osmotic stresses, and indicate that it is a complex process involving transient changes in water content and sustained changes in electrolyte and organic osmolyte contents. Appreciation of the nature of the adaptation process, and conversely the deadaptation processes that occur after recovery from hypoosmolality and hyperosmolality, enables a better understanding of the marked variations in neurological sequelae that characterize hyperosmolar and hypoosmolar states, and provides a basis for more rational therapies.     

Aquaporins and cell migration

Aquaporin (AQP) water channels are expressed primarily in cell plasma membranes. In this paper, we review recent evidence that AQPs facilitate cell migration. AQP-dependent cell migration has been found in a variety of cell types in vitro and in mice in vivo. AQP1 deletion reduces endothelial cell migration, limiting tumor angiogenesis and growth. AQP4 deletion slows the migration of reactive astrocytes, impairing glial scarring after brain stab injury. AQP1-expressing tumor cells have enhanced metastatic potential and local infiltration. Impaired cell migration has also been seen in AQP1-deficient proximal tubule epithelial cells, and AQP3-deficient corneal epithelial cells, enterocytes, and skin keratinocytes. The mechanisms by which AQPs enhance cell migration are under investigation. We propose that, as a consequence of actin polymerization/depolymerization and transmembrane ionic fluxes, the cytoplasm adjacent to the leading edge of migrating cells undergoes rapid changes in osmolality. AQPs could thus facilitate osmotic water flow across the plasma membrane in cell protrusions that form during migration. AQP-dependent cell migration has potentially broad implications in angiogenesis, tumor metastasis, wound healing, glial scarring, and other events requiring rapid, directed cell movement. AQP inhibitors may thus have therapeutic potential in modulating these events, such as slowing tumor growth and spread, and reducing glial scarring after injury to allow neuronal regeneration.     

Three-dimensional confocal morphometry - a new approach for studying dynamic changes in cell morphology in brain slices

Pathological states in the central nervous system lead to dramatic changes in the activity of neuroactive substances in the extracellular space, to changes in ionic homeostasis and often to cell swelling. To quantify changes in cell morphology over a certain period of time, we employed a new technique, three-dimensional confocal morphometry. In our experiments, performed on enhanced green fluorescent protein/glial fibrillary acidic protein astrocytes in brain slices in situ and thus preserving the extracellular microenvironment, confocal morphometry revealed that the application of hypotonic solution evoked two types of volume change. In one population of astrocytes, hypotonic stress evoked small cell volume changes followed by a regulatory volume decrease, while in the second population volume changes were significantly larger without subsequent volume regulation. Three-dimensional cell reconstruction revealed that even though the total astrocyte volume increased during hypotonic stress, the morphological changes in various cell compartments and processes were more complex than have been previously shown, including swelling, shrinking and structural rearrangement. Our data show that astrocytes in brain slices in situ during hypotonic stress display complex behaviour. One population of astrocytes is highly capable of cell volume regulation, while the second population is characterized by prominent cell swelling, accompanied by plastic changes in morphology. It is possible to speculate that these two astrocyte populations play different roles during physiological and pathological states.     

Neural control of hepatic osmolytes and parenchymal cell hydration

Liver cell function is strongly influenced by cell hydration and osmolyte content via osmosensing and osmosignaling pathways. Electrical stimulation of hepatic nerves increases the hepatocellular hydration state via an alpha-adrenergic mechanism, promotes taurine release from parenchymal cells and myo-inositol release from hepatic stellate cells. Although changes in liver cell hydration and osmolyte content are known regulators of liver cell function, the functional relevance and integration of nerve-stimulation-dependent alterations of liver cell volume and osmolyte content in the overall hepatic response to towards signals from the nervous system remains to be established.     

Terbutaline-induced triphasic changes in volume of rat alveolar type II cells: the role of cAMP

Changes in the volume of rat alveolar type II cells (AT-II cells) induced by terbutaline, a beta(2)-agonist, were measured using video-enhanced contrast microscopy. The changes consisted of three phases: initial cell shrinkage, cell swelling, and gradual cell shrinkage. The initial cell shrinkage was Ca(2+)-dependent and was inhibited by quinine (a K+ channel blocker). The subsequent cell swelling was cAMP-dependent and was inhibited by amiloride (a Na+ channel blocker). The final cell shrinkage was cAMP-dependent and was inhibited by 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB, a Cl- channel blocker). Thus, terbutaline-induced cell volume changes were regulated by both Ca2+ and cAMP. Accumulation of cAMP alone, however, induced the Ca2+ -dependent cell shrinkage of AT-II cells and H-89 (a PKA inhibitor) inhibited terbutaline-induced cell volume changes. This suggests that cAMP accumulation stimulates the Ca2+ signal during terbutaline stimulation. In conclusion, terbutaline stimulates not only Na+ influx, but also K+ and Cl- release mediated via cAMP accumulation in rat AT-II cells, which induces the triphasic cell volume changes.     

Does anoxia induce cell swelling in carp brains? In vivo MRI measurements in crucian carp and common carp

Although both common and crucian carp survived 2 h of anoxia at 18 degrees C, the response of their brains to anoxia was quite different and indicative of the fact that the crucian carp is anoxia tolerant while the common carp is not. Using in vivo T(2) and diffusion-weighted magnetic resonance imaging (MRI), we studied anoxia induced changes in brain volume, free water content (T(2)), and water homeostasis (water diffusion coefficient). The anoxic crucian carp showed no signs of brain swelling or changes in brain water homeostasis even after 24 h except for the optic lobes, where cellular edema was indicated. The entire common carp brain suffered from cellular edema, net water gain, and a volume increase (by 6.5%) that proceeded during 100 min normoxic recovery (by 10%). The common carp recovered from this insult, proving that the changes were reversible and suggesting that the oversized brain cavity allows brain swelling during energy deficiency without a resultant increase in intracranial pressure and global ischemia. It is tempting to suggest that this is a function of the large brain cavity seen in many ectothermic vertebrates.     

肾上腺皮质激素对水通道蛋白表达的调节

肾上腺皮质激素参与水盐代谢的调节,盐皮质激素和糖皮质激素调节上皮细胞水通道蛋白 (Aquaporins,AQPs)的表达。盐皮质激素可使肾脏AQP3表达上调,对肾脏的AQP2表达依据刺激的时间不 同而表达不同,但对AQP1没有影响。糖皮质激素对不同组织器官AQPs的影响不尽相同。肾上腺皮质激素 对AQPs的调节作用在某些水代谢紊乱疾病的诊断与治疗方面可能有着广阔的应用前景。     

Osmometric and water-transporting properties of guinea pig cardiac myocytes

To elucidate the mechanism of water flux across heart cell membranes, osmotically induced volume changes and sarcolemmal water permeability were evaluated in isolated guinea pig ventricular myocytes by videomicroscopic measurements of cell surface dimensions. Superfusion with anisosmotic solution (0.5-4 times normal osmolality) caused a rapid (lt;3 min to new steady state) and reversible cell swelling or shrinkage mainly because of proportional changes in cell width and thickness. The van't Hoff relationship between relative cell volume and the reciprocal of relative osmolality was linear and predicted an apparent osmotically dead space of approximately 35% cell volume. The osmotic water permeability coefficient (P(f)) measured from the time course of cell swelling/shrinkage was approximately 22 microm.s(-1) at 35 degrees C. Arrhenius activation energy (E(a)), a measure of the energy barrier to water flux, was approximately 3.8 kcal.mol(-1) between 11 and 35 degrees C; this value is equivalent to E(a) for free-water diffusion in bulk solution ( approximately 4 kcal.mol(-1)). Treatment with 0.1 mM Hg(2+), a sulfhydryl-oxidizing reagent, reduced P(f) by approximately 90%, and the sulfhydryl-reducing reagent dithiothreitol (10 mM) antagonized the inhibitory action of Hg(2+). E(a) measured from Hg(2+)-treated myocytes (12.3 kcal.mol(-1)) was in the range of that for diffusional water movement through the lipid bilayers (>10 kcal.mol(-1)). Although the observed P(f) is small in magnitude, both the low E(a) and the sulfhydryl-related modifications of P(f) are characteristic of channel-mediated water transport. These data suggest that water channels form a major conduit for water crossing the sarcolemma of guinea-pig heart cells.     

Rapid and slow swelling during hypoxia in the CA1 region of rat hippocampal slices.

The role of swelling in hypoxic/ischemic neuronal injury is incompletely understood. We investigated the extent and time course of cell swelling during hypoxia, and recovery of cell volume during reoxygenation, in the CA1 region of rat hippocampal slices in vitro. Cell swelling was measured optically and compared with simultaneous measurements of the extracellular DC potential, extracellular [K+], and synaptic transmission in the presence and absence of hypoxic depolarization. Hypoxia-induced swelling consisted of rapid and/or slow components. Rapid swelling was observed frequently and always occurred simultaneously with hypoxic depolarization. Additionally, rapid swelling was followed by a prolonged phase of swelling that was approximately 15 times slower. Less frequently, slow swelling occurred independently, without either hypoxic depolarization or a preceding rapid swelling. For slices initially swelling rapidly, recovery of both cell volume and the slope of field excitatory postsynaptic potentials were best correlated with the duration of hypoxia (r = 0.77 and 0.87, respectively). This was also the case for slices initially swelling slowly (r = 0.70 and 0.58, respectively). In contrast, the degree of recovery of cell volume was the same at 30 or 60 min of reoxygenation, indicating that prolonging the duration of reoxygenation within these limits was ineffective in improving recovery. Spectral measurements indicated that the hypoxia-induced changes in light transmittance were related to changes in cell volume and not changes in the oxidation state of mitochondrial cytochromes. The persistent impairment of synaptic transmission in slices swelling slowly (i.e., without hypoxic depolarization) indicates that swelling may play a role in this injury and that hypoxic depolarization is not required. Additionally, the correlation between the degree of recovery of cell volume and the degree of recovery of synaptic transmission during reoxygenation supports a role for swelling in hypoxic neuronal injury.     

Migration of transformed renal epithelial cells is regulated by K+ channel modulation of actin cytoskeleton and cell volume

Migration of transformed renal epithelial (MDCK-F) cells depends on the polarized activity of a Ca²⁺-sensitive K⁺ channel (IK channel; Pflügers Arch 432:R87–R93, 1996). This study was aimed at elucidating the functional link between the IK channel and the actin cytoskeleton which is required for cell locomotion. We monitored migration of MDCK-F cells with video microscopy, quantified filamentous actin with phalloidin binding, and measured the intracellular Ca²⁺ concentration ([Ca²⁺]_i) with the fluorescent dye fura-2/AM. We compared the effects of IK channel activation or inhibition with those of hypotonic swelling or hypertonic shrinkage. IK channel inhibition with charybdotoxin (CTX) or cell swelling (omission of up to 50 mmol/l NaCl) as well as IK channel activation with 1-ethyl-2-benzimidazolinone (1-EBIO) or cell shrinkage (addition of up to 100 mmol/l mannitol) reduce the rate of migration dose-dependently by up to 80%, i.e., to the same extent as cytochalasin D. Inhibition of migration is accompanied either by actin depolymerization (CTX and cell swelling) or by actin polymerization (1-EBIO and cell shrinkage). Changes of migration and phalloidin binding induced by CTX and cell swelling or by 1-EBIO and cell shrinkage, respectively, are linearly correlated with each other. CTX and cell swelling elicit a rise of [Ca²⁺]_i whereas 1-EBIO and cell shrinkage induce a slight decrease of [Ca²⁺]_i in most MDCK-F cells. Taken together IK-channel-dependent perturbations of cell volume and anisotonicity elicit virtually identical effects on migration, actin filaments and [Ca²⁺]_i. We therefore suggest that cell volume – possibly via [Ca²⁺]_i– is the link between IK channel activity, actin filaments and migration. We propose a model for how temporal and local changes of cell volume can support the migration of MDCK-F cells. Received: 28 January 1999 / Received after revision: 15 April 1999 / Accepted: 16 April 1999     

Two different mechanisms underlie reversible, intrinsic optical signals in rat hippocampal slices

Intrinsic optical signals (IOSs) induced by synaptic stimulation and moderate hypotonic swelling in brain tissue slices consist of reduced light scattering and are usually attributed to cell swelling. During spreading depression (SD), however, light-scattering increases even though SD has been shown to cause strong cell swelling. To understand this phenomenon, we recorded extracellular voltage, light transmission (LT), which is inversely related to light scattering, and interstitial volume (ISV) simultaneously from the same site (stratum radiatum of CA1) in both interface and submerged hippocampal slices. As expected, moderate lowering of bath osmolarity caused concentration-dependent shrinkage of ISV and increase in LT, while increased osmolarity induced opposite changes in both variables. During severe hypotonia, however, after an initial increase of LT, the direction of the IOS reversed to a progressive decrease in spite of continuing ISV shrinkage. SD caused by hypotonia, by microinjection of high-K(+) solution, or by hypoxia, was associated with a pronounced LT decrease, during which ISV shrinkage indicated maximal cell swelling. If most of the extracellular Cl(-) was substituted by the impermeant anion methylsulfate and also in strongly hypertonic medium, the SD-related decrease in LT was suppressed and replaced by a monotonic increase. Nevertheless, the degree of ISV shrinkage was similar in low and in normal Cl(-) conditions. The optical signals and ISV changes were qualitatively identical in interface and submerged slices. We conclude that there are at least two mechanisms that underlie reversible optical responses in hippocampal slices. The first mechanism underlies light-scattering decrease (hence enhancing LT) when ISV shrinks (cell swelling) under synaptic stimulation and mild hypotonia. Similarly, as result of this mechanism, expansion of ISV (cell shrinkage) during mild hypertonia leads to an increased light scattering (and decreased LT). Thus optical signals associated with this first mechanism show expected cell-volume changes and are linked to either cell swelling or shrinkage. A different mechanism causes the light-scattering increase (leading to a LT decrease) during severe hypotonia and various forms of SD but with a severely decreased ISV. This second mechanism may be due to organelle swelling or dendritic beading but not to cell-volume increase. These two mechanisms can summate, indicating that they are independent in origin. Suppression of the SD-related light-scattering increase by lowering [Cl(-)](o) or severe hypertonia unmasks the underlying swelling-related scattering decrease. The simultaneous IOS and ISV measurements clearly distinguish these two mechanisms of optical signal generation.     

The involvement of caspases in the CD95(Fas/Apo-1)- but not swelling-induced cellular taurine release from Jurkat T-lymphocytes

Following a delay of 45 min, stimulation of the CD95 (Fas/Apo-1)-receptor in Jurkat T-lymphocytes leads to the release of the osmolyte taurine, an event coinciding with apoptotic cell shrinkage. The present study has been performed to elucidate the cellular mechanisms involved in CD95-induced taurine release as compared to swelling-induced taurine release, and to explore whether taurine modifies apoptotic DNA fragmentation and cell shrinkage. Taurine release stimulated by osmotic cell swelling is insensitive to the tyrosine kinase inhibitor herbimycin A and the caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD) but is blunted in the absence of extracellular Ca2+. Conversely, the Ca2+ ionophore ionomycin stimulates taurine release. However, the taurine release following CD95 stimulation is not paralleled by an increase of cytosolic Ca2+ and not inhibited by complexation of extracellular Ca2+. None of herbimycin A, the phosphatase inhibitor vanadate, spingomyelinase or Lck56 deficiency prevent CD95-induced taurine release. In contrast, the caspase inhibitor zVAD, but not the caspase inhibitor Ac-Tyr-Val-Ala-Asp-chloromethylketone (YVAD), almost abolishes CD95-induced taurine release. Both caspase inhibitors blunt CD95-induced cell shrinkage and DNA fragmentation, zVAD being more effective than YVAD. Preloading of the cells with 40 mM taurine but not with 40 mM mannitol significantly inhibits CD95-induced DNA fragmentation (by 28%) and apoptotic cell shrinkage (by 25%). In conclusion, CD95-receptor triggering leads to caspase-dependent stimulation of cellular taurine release, which facilitates, but is not sufficient for, the triggering of apoptotic DNA fragmentation and cell shrinkage.     

Water permeability through aquaporin-4 is regulated by protein kinase C and becomes rate-limiting for glioma invasion

Glial-derived tumors, gliomas, are highly invasive cancers that invade normal brain through the extracellular space. To navigate the tortuous extracellular spaces, cells undergo dynamic changes in cell volume, which entails water flux across the membrane through aquaporins (AQPs). Two members of this family, AQP1 and AQP4 are highly expressed in primary brain tumor biopsies and both have a consensus phosphorylation site for protein kinase C (PKC), which is a known regulator of glioma cell invasion. AQP4 colocalizes with PKC to the leading edge of invading processes and clustered with chloride channel (ClC2) and K⁺–Cl⁻ cotransporter 1 (KCC1), believed to provide the pathways for Cl⁻ and K⁺ secretion to accomplish volume changes. Using D54MG glioma cells stably transfected with either AQP1 or AQP4, we show that PKC activity regulates water permeability through phosphorylation of AQP4. Activation of PKC with either phorbol 12-myristate 13-acetate or thrombin enhanced AQP4 phosphorylation, reduced water permeability and significantly decreased cell invasion. Conversely, inhibition of PKC activity with chelerythrine reduced AQP4 phosphorylation, enhanced water permeability and significantly enhanced tumor invasion. PKC regulation of AQP4 was lost after mutational inactivation of the consensus PKC phosphorylation site S180A. Interestingly, AQP1 expressing glioma cells, by contrast, were completely unaffected by changes in PKC activity. To demonstrate a role for AQPs in glioma invasion in vivo, cells selectively expressing AQP1, AQP4 or the mutated S180A-AQP4 were implanted intracranially into SCID mice. AQP4 expressing glioma cells showed significantly reduced invasion compared to AQP1 and S180 expressing tumors as determined by quantitative stereology, consistent with a differential role for AQP1 and AQP4 in this process.     

AQP1 mediates water transport in the carotid body

In this study, we explored the presence of aquaporins (AQPs), a family of membrane water channel proteins, in carotid body (CB) type I chemoreceptor cells. The CB is a polymodal chemoreceptor whose major function is to detect changes in arterial O₂ tension to elicit hyperventilation during hypoxia. The CB has also been proposed to function as a systemic osmoreceptor, thus we hypothesized that the presence of AQPs in type I cell membrane may confer higher sensitivity to osmolarity changes and hence accelerate the activation of chemoreceptor cells. We detected the expression of AQP1, AQP7, and AQP8 in the CB and confirmed the location of AQP1 in type I cells. We have also shown that inhibition of AQP1 expression clearly reduced type I cell swelling after a hyposmotic shock, demonstrating that AQP1 has a major contribution in transmembrane water movement in these chemoreceptor cells. Interestingly, CB AQP1 expression levels change during postnatal development, increasing during the first postnatal weeks as the organ matures. In conclusion, in this study, we report the novel observation that AQPs are expressed in the CB. We also show that AQP1 mediates water transport across the cell membrane of type I cells, supporting the contribution of this protein to the osmoreception function of the CB.     

Volume regulation by Necturus gallbladder: apical Na+-H+ and Cl(-)-HCO-3 exchange

Necturus gallbladder epithelial cells exhibited volume regulatory swelling when exposed to a hypertonic mucosal bathing solution. The initial, osmotically induced shrinkage was followed by a rapid increase in cell volume back to the control value despite continuing hypertonicity of the mucosal perfusate. This volume regulatory increase occurred by osmotic water flow accompanying the transient cellular uptake of NaCl from the mucosal bathing solution. Volume regulatory increase required Na+ and Cl- in the mucosal bath; it was inhibited by amiloride or 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid but not by bumetanide or ouabain. The K1/2 for Na+ was 2.8 mM, the K1/2 for Cl- was 1.9 mM, and maximum velocity of fluid flow into the cell for both ions was greater than 10 x 10(-6) cm/s. Both volume regulatory increase and transepithelial fluid absorption involve NaCl flux across the apical membrane into the cells, but the nature of the NaCl fluxes differ in the two processes. During volume regulatory increase NaCl enters the cells by parallel Na+-H+ and Cl(-)-HCO-3 exchanges, whereas during transepithelial fluid absorption NaCl enters the cell by the coupled flux of NaCl.     

水通道蛋白与神经系统疾病关系的研究进展

20世纪80年代,美国学者Peter Agre发现质膜上有构成水通道的膜蛋白,这种膜蛋白被命名为水孔蛋白或水通道蛋白(AQP)。水通道蛋白的发现,揭示了水可快速穿膜流动的机制,而且通过研究发现,水通道蛋白与人体多种疾病的发生有关,如神经系统、肾脏、肠道、眼以及耳鼻喉等的疾病。近年来,人们针对水通道蛋白在各种疾病中的生理、病理作用进行了大量的研究。我们着眼于水通道蛋白与神经系统疾病间的关系,重点对在神经系统疾病中起关键作用的AQP-1和AQP-4的研究新进展进行综述。     

Regulatory volume increase after hypertonicity- or vasoactive-intestinal-peptide-induced cell-volume decrease in small-intestinal crypts is dependent on Na+-K+-2Cl− cotransport

The volume of intact crypts isolated from guinea-pig small intestine has been measured to assess the capacity of the cells to regulate their volume after hypertonic shock or vasoactive-intestinal-peptide (VIP)-induced shrinkage. Crypts exposed to anisotonic medium initially behave as perfect osmometers. Continued exposure to a hypertonic (400 mosmol/l) medium was followed by regulatory volume increase (RVI), which led to complete volume recovery in about 20 min. VIP produced a volume reduction, attributed to KCl loss through channels activated by the secretagogue, without any recovery during exposure to the polypeptide. Removal of VIP led to an increase of cellular volume towards control levels. This volume recovery after secretagogue-induced shrinkage is termed SVI. Both RVI and SVI were abolished by removal of Na⁺ or Cl^– from the bathing solution, by addition of the loop diuretic bumetanide (1 M), but not by addition of ethylisopropylamiloride (10 M) or amiloride (1 mM). Cell shrinkage was also observed when tonicity was increased by addition of 100 mM NaCl or 200 mM D-mannitol, but RVI was seen only when NaCl was the added osmolyte. The ion dependence, pharmacological sensitivity and thermodynamic considerations of these effects are consistent with the operation of a Na⁺-K⁺-2Cl^– cotransport mechanism activated by cell shrinkage and the secretagogue action of VIP.     

Morphometric study of cardiac muscle: the problem of tissue shrinkage

Comparison of data from morphometric studies dealing with the heart is complicated by the fact that little information dealing with cell size changes during tissue processing is available. To investigate these changes, isolated cardiac myocytes were adhered to glass cover slips of Sykes Moore chambers and photographed after each step of processing for transmission electron microscopy. Six different experiments with a minimum of 10 cells each were followed through the entire procedure after fixation with isoosmolar glutaraldehyde. Cellular dimension changes were determined by tracing individual isolated myocytes after each step of the procedure with a sonic digitizer. Significant cell volume changes occurred after osmium (16 per cent swelling), postosmium wash (10 per cent swelling), and uranyl acetate (25 per cent shrinkage). Hypertonic aldehyde solutions resulted in cellular shrinkage during fixation not found with isotonic solutions. Changes in cell cross-sectional area rather than length were largely responsible for altered cell volumes during any given phase of processing. The results indicate that, although cell volume changes occur during processing, final cell dimensions of embedded cells were not different from unfixed cells. In whole tissue blocks, inclusion of propylene oxide in the procedure resulted in tissue shrinkage which was not observed in isolated myocytes, suggesting that different tissue components react in a variable manner to propylene oxide. After each of the other steps in processing, tissue blocks reacted in a similar manner to the isolated myocytes.     

脑缺血合并高脂血症模型大鼠脑组织的病理改变

背景：大多数脑缺血是在高血压、高脂血症、糖尿病等基础病变条件下发生的。因此,构建高脂血症复合脑缺血大鼠模型,研究基础性病变对脑缺血的影响具有重要意义。 目的：观察高脂血症复合脑缺血大鼠模型脑组织病理学改变,及其高脂血症病理因素对脑缺血的影响。 方法：实验以高脂饲料喂养大鼠制备高脂血症大鼠模型,然后线栓法制备局灶性脑缺血大鼠模型,建模成功后3,7 d,采用TTC染色的方法,观察各组大鼠脑组织缺血部位体积,苏木精-伊红染色观察各组大鼠脑组织缺血边缘区组织病理学改变,透射电镜观察各组大鼠脑组织缺血边缘区细胞超微结构改变。 结果与结论：TTC染色结果显示高脂+脑缺血7 d组大鼠的脑缺血部位体积明显减小。苏木精-伊红染色结果显示所有脑缺血模型都呈典型的缺血性改变,脑缺血7 d的小胶质细胞数量比3 d的明显减少,高脂+脑缺血7d相对于3 d的变化更明显。超微结构显示所有脑缺血模型的神经元和胶质细胞核膜皱缩,线粒体嵴基本完全消失,内皮细胞线粒体减少,神经突触的突触小泡大部分溶解,缺血7 d,尤其是高脂+脑缺血7 d的上述损伤减轻,神经元变性、坏死减少,线粒体损伤恢复,线粒体嵴也明显增多,神经突触的突触小泡明显恢复。说明高脂血症促进了脑缺血损伤的恢复,其原因可能是高脂血症因素激活了体内某种保护机制。