Formalin Perfusion Research Articles

<i>Curcuma longa</i> renal historestorative effects on sildenafil induced nephrotoxicity among male albino rats

Background: Sildenafil is a phosphodiesterase type 5 inhibitor mostly used in management of erectile dysfunction and pulmonary hypertension. On the other hand, Curcuma longa is herbal plant that is mostly used in diets among the Asian and African population and commonly used in treatment of respiratory, renal and dermatological diseases.
 Methods: A post-test only true experimental study design was utilized with 25 Albino rats grouped as follows: negative control, positive control (sildenafil 1µg/g bwt), low dose Curcuma longa (38.75mg/kg), medium dose Curcuma longa (77.5mg/kg) and high dose Curcuma longa (155mg/kg) each having 5 rats. Induction of nephrotoxicity using sildenafil was done for 15 days with an interim sacrifice for negative and positive control groups done on day 15. Experimental groups were sacrificed on day 22 after receiving Curcuma longa at respective doses. Blood samples for renal function tests were obtained on day 1, 15 and 22. Enblock harvesting of the kidneys after euthanizing the rats with concentrated CO2 and 10% neutral buffered formalin perfusion was done on day 15 and 22 then fixed in 10% neutral buffered formalin for 24hours. The kidneys were then processed for Hematoxylin and Eosin staining and photomicrographs were taken using Olympus light microscope fitted with LABOMED ivu 3100 digital camera.
 Results: In sildenafil induced nephrotoxicity group; glomerulus was distorted, dilated bowman`s space, dilated renal tubules and necrotic epithelial cells. Minimal histological changes were observed in low Curcuma longa dose group while in medium and high Curcuma longa dose groups; the glomerulus had well defined margins, bowman`s space reduced in size, epithelial cells appeared normal.
 Conclusion: It can be concluded that medium and high dose Curcuma longa have kidney historestorative effects in sildenafil induced nephrotoxicity among male albino rats.

Settlement of Stenotic Site and Enhancement of Risk Factor Load for Atherosclerosis in Left Anterior Descending Coronary Artery by Myocardial Bridge.

The aim of this study was to investigate the influence of a myocardial bridge (MB) on atherosclerosis development in the left anterior descending artery of the normal heart and the importance of traditional risk factors (RFs). An additional objective was to determine the correlation between intimal thickening and luminal narrowing. The left anterior descending artery from 150 autopsied hearts was treated with formalin perfusion fixation, and each left anterior descending artery was serially cross-sectioned. The intima-media and luminal stenosis ratios were examined using computer-assisted histomorphometry. The luminal stenosis ratio was closely correlated with the intima-media ratio (r=0.792; P<0.001). When an MB was present, the luminal stenosis ratios proximal to the MB in the RF (+) group were significantly greater than those in the RF (-) group (P=0.022 by a multiple comparison test), but there were no differences between the RF (+) and RF (-) groups when an MB was absent. In addition, the site of the greatest stenosis in the MB (+) RF (+) group was 2.5 cm proximal to the MB entrance. Multivariate analyses indicated that age was an independent factor for luminal stenosis ratios ≥50% and 60% (P=0.002 and 0.029, respectively). Furthermore, the presence of an MB plus RFs was an independent factor for a luminal stenosis ratio ≥70% (P=0.037). An MB enhances left anterior descending artery atherosclerosis development at a site proximal to the MB entrance, particularly in subjects who have some RFs.

The use of a cerebral perfusion and immersion–fixation process for subsequent white matter dissection

BackgroundThe Klingler's method for white matter dissection revolutionized the study of deep cerebral anatomy. Although this technique made white matter dissection more feasible and widely used, it still presents some intrinsic limitations. New methodWe evaluated the quality of different methods for specimen preparation based on an intra-carotidal formalin perfusion fixation process. Ten post-mortem human hemispheres were prepared with this method and dissected in a stepwise manner. ResultsThe homogeneous and rapid fixation of the brain allowed documentation of several fine additional anatomical details. Intra-cortical white matter terminations were described during the first stage of dissection on each specimen. No limitations were encountered during dissection of the major associative bundles. On the contrary, the quality of the fixation of the specimens made it possible to isolate them en bloc. One of the most complex and deep bundles (accumbo-frontal fasciculus) was dissected without technical limitations. Deep vascular structures were very well preserved and dissected within the white matter until their sub-millimetric terminations. Comparison with existing methodShort time for preparation, a more homogeneous fixation, no technical limitation for a detailed description of superficial and deep white matter anatomy, the possibility to dissect with a single technique the fibre organization and the white matter vascular architecture are the advantages reported with the perfusion fixation. ConclusionThese results provide encouraging data about the possibility to use a perfusion fixation process, which may help in improving the quality of white matter dissection for research, didactic purposes and surgical training.

A method for generating pulmonary neutrophilia using aerosolized lipopolysaccharide.

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models of ALI are key in enhancing our understanding of disease pathogenesis. Lipopolysaccharide (LPS) derived from gram positive bacteria induces neutrophilic inflammation in the airways and lung parenchyma of mice. Efficient pulmonary delivery of compounds such as LPS is, however, difficult to achieve. In the approach described here, pulmonary delivery in mice is achieved by challenge to aerosolized Pseudomonas aeruginosa LPS. Dissolved LPS was aerosolized by a nebulizer connected to compressed air. Mice were exposed to a continuous flow of LPS aerosol in a Plexiglas box for 10 min, followed by 2 min conditioning after the aerosol was discontinued. Tracheal intubation and subsequent bronchoalveolar lavage, followed by formalin perfusion was next performed, which allows for characterization of the sterile pulmonary inflammation. Aerosolized LPS generates a pulmonary inflammation characterized by alveolar neutrophilia, detected in bronchoalveolar lavage and by histological assessment. This technique can be set up at a small cost with few appliances, and requires minimal training and expertise. The exposure system can thus be routinely performed at any laboratory, with the potential to enhance our understanding of lung pathology.

Superselective Intraophthalmic Artery Chemotherapy in a Nonhuman Primate Model

We describe the histopathologic findings in a nonhuman primate (NHP) model of superselective intraophthalmic artery chemotherapy (SSIOAC), detailing ocular and orbital vascular adverse effects. To further document, using comprehensive ocular and orbital histopathology, previously reported toxic effects observed with real-time ophthalmoscopy during SSIOAC in a NHP model. Comparative interventional case series. Preclinical trial approved under the guidelines of the Institutional Animal Care and Utilization committee. Six adult male rhesus macaques (Macacca mulatta). The right eye of each NHP was treated with 3 cycles of SSIOAC using either melphalan (5 mg/30 mL) or carboplatin (30 mg/30 mL). Both eyes in each animal were enucleated 6 hours after the final procedure, before euthanasia and formalin perfusion of the NHP; we then performed orbital dissection of the arterial vasculature and optic nerves. Histopathologic examination of the eyes, optic nerves, and orbital vessels of the 6 treated NHPs. We found leukostasis with retinal arteriole occlusion in all treated eyes. Retinal endothelial cells stained positive for 2 inflammatory markers, intercellular adhesion molecule 1 and interleukin 8. Transmission electron microscopy revealed occlusion of the retinal vessels with ultrastructural changes in the endothelial cells and surrounding pericytes. Additional findings included nerve fiber layer infarcts, central retinal artery thrombosis, hypertrophy and occlusion of choroidal arteries with disruption of the internal elastic lamina, patchy choroidal inflammation, and birefringent intravascular foreign bodies. Orbital findings included ophthalmic artery and central retinal artery wall dissection, fracturing of the internal elastic lamina, intimal hyperplasia, and eyelid vessel damage. Optic nerves displayed hemorrhage, leukostasis, and foreign body crystallization. Control eyes, optic nerves, and orbital vessels were normal. Histopathologic examination of our nonhuman primate model for SSIOAC revealed significant toxic effects in the ocular and orbital vasculature. These findings substantiate previous observations with real-time retinal imaging and parallel reported vascular toxic effects in children with retinoblastoma treated with SSIOAC.

A New Liver Explant Fixation Technique

To the Editor.—Surgical intervention, including orthotopic liver transplantation, is a common modality used in the treatment of hepatocellular carcinoma. Accurate pathologic information is important to clinicians for appropriate staging and prognostication of such patients, particularly the identification of tumor multifocality. To identify all potentially neoplastic nodules, a meticulous gross examination is necessary.The gross examination of liver explants typically involves “bread-loafing” the specimen into slices as thin as practicably possible, ideally 0.5 cm or less in thickness. However, obtaining thin, uniform slices may be challenging in large unfixed hepatectomy specimens. In clinical practice, irregular slices thicker than 0.5 cm are frequently obtained, which increases the frequency of missed lesions, particularly small lesions. In addition, correlation with preoperative imaging studies becomes increasingly difficult in specimens cut irregularly.Formalin fixation firms the gross liver texture and improves the cutting process, making possible more uniform slices as compared with unfixed liver specimens. Ideally, formalin fixation should occur evenly throughout the entire liver parenchyma. Immersing intact specimens in formalin is generally inadequate for this purpose as formalin permeates through tissue slowly, resulting in a thin rind of fixed liver surrounding an unfixed core. To achieve uniform fixation, a group at Mayo Clinic (Rochester, Minn) developed a perfusion process that delivers formalin throughout the vasculature of the entire specimen1 via a continuous-perfusion pump for 3 days. This process is slow, labor intensive, and requires specialized equipment.An alternative technique that is quick, easy to use, inexpensive, and effective is herein described. It involves the use of an appropriately sized Foley catheter to cannulate the large vessels of the explanted liver (Figure 1). Once cannulated, the balloon cuff is inflated to prevent backflow. A catheter tip 60-mL syringe is then used to perfuse formalin into the hepatic vasculature. The entire process can be performed in less than 30 minutes. Because high pressures can easily be produced during perfusion that may inadvertently produce artifactual damage it is recommended that the force of only one hand be used. Good fixation results have been achieved with as little as 200 mL of formalin per large vessel (Figure 2). Following formalin perfusion, the intact specimen is floated in a bath of formalin overnight.In our experience, cannulation and perfusion of only the hepatic veins suffices to achieve adequate fixation. Portal vein cannulation is generally unnecessary but may be performed if anatomy permits. Hepatic artery cannulation is technically challenging and is of negligible added value.This technique results in a well-fixed, firm specimen that is easily sliced at the requisite 0.5-cm intervals, making possible a meticulous gross examination that aids in the identification of even small liver nodules.

Immunohistochemical detection of induced expression of wild-type p53 tumor suppressor protein in the livers of rats treated with diethylnitrosamine.

Paraffin sections of formalin-perfused rat livers were stained immunohistochemically for p53. In livers from untreated rats, no p53 expression was observed. p53 expression was induced in a response to treatment with diethylnitrosamine 24 h prior to sacrifice. Staining for p53 was localized in the nucleus of perivenous hepatocytes. In serial sections p53-immunopositive areas were found to co-localize with increased expression of TUNEL-positive cells. Without formalin perfusion, the staining for p53 was uneven and often barely detectable. Perfusion with saline prior to formalin resulted in a rapid decrease in the detectability of p53, indicating rapid degradation of this protein under these conditions. We conclude that rapid fixation by formalin perfusion increases the detectability of p53 by immunohistochemical staining. This provides a convenient procedure for studying the response of wild-type p53 in rodent liver. This procedure is also suitable for in situ investigations on the degradation of p53 protein stabilized by DNA damage.

Effects of ethanol in traumatic brain injury.

The effects of ethanol intoxication on brain injury and cerebral blood flow (CBF) were investigated in a porcine fluid-percussion model of traumatic brain injury (TBI). Immature swine, under halothane anesthesia (1%), had a TBI delivered with a fluid-percussion device. The experimental group (n = 10) received ethanol (3.5 gm/kg) via gastric tube followed in 1 h by TBI. Two groups of control animals received normal saline and TBI (n = 10) or ethanol and no TBI (n = 5). Mean arterial blood pressure (MAP), intracranial pressure (ICP), arterial blood gases, and serum lactate were monitored for 3 h after the injury. CBF was measured with radiolabelled 15-micron diameter microspheres. Neuropathologic changes were evaluated and graded after formalin perfusion and brain removal at 3 h postinjury. The ethanol level 60 min post-head injury was 198 +/- 70 (SD) mg/dL in the ethanol+TBI group. At 90 min postinjury and thereafter, ethanol+TBI animals compared with TBI only animals had significantly lower MAP (63 +/- 26 mmHg vs 91 +/- 15 mmHg) and lower cerebral perfusion pressure (50 +/- 25 mmHg vs 78 +/- 15), and at 180 min postinjury, lower CBF (87 +/- 37% vs 62 +/- 79% of preinjury levels). Ethanol+TBI animals had higher blood lactates (28 +/- 11 mg/dL vs 13 +/- 6 mg/dL) than TBI only animals. Ethanol+TBI animals also had significantly longer postinjury apneas (11 +/- 8 min vs 0.6 +/- 0.4 min), with three of ten ethanol-treated animals never recovering spontaneous respiration. Ethanol intoxication produced hemodynamic and respiratory changes, which may have a deleterious effect on outcome and mortality after brain injury.

Distribution of ‘Non-specific’ cholinesterase-containing neurons in the dorsal thalamus of the rat

This report describes the distribution of histochemically identified 'non-specific' cholinesterase (ChE)-containing neurons in the dorsal thalamus of the rat. Juvenile or young adult Long-Evans or Sprague-Dawley rats were sacrificed by formalin perfusion. Some animals received systemic injections of 1.5-2.0 mg/kg DFP 4-24 h prior to sacrifice. Separate series of 50 micron frozen sections were processed for cholinesterase histochemistry using acetylthiocholine, butyrylthiocholine, or propionylthiocholine as substrates. Adjacent sections processed with each of the 3 substrates allowed comparison of the distributions of neurons containing the histochemical reaction products. Neurons containing moderate to high concentrations of ChE reaction product were found in 3 distinct regions of the dorsal thalamus. First, neurons staining intensely for ChE were found in a cluster that corresponds to the thalamic reuniens nucleus. Second, a cluster of neurons staining intensely for ChE was found in a region that included the lateral part of the central lateral nucleus and extended laterally into the ventral-lateral part of the lateral dorsal nucleus. Third, moderate ChE staining was observed in the neurons of the anterior dorsal nucleus. Of these regions, only the anterior dorsal nucleus shows moderate to high levels of acetylcholinesterase. The function of ChE in normal brain function is unknown. It is particularly interesting, however, that the thalamic nuclei containing ChE-positive neurons send thalamocortical projections to the medial limbic cortex, including cingulate, retrosplenial and subicular cortices.

Fiber bundle direction in the mammalian heart. An extension of the "nested shells" model.

The orientation of the muscle fiber bundles within the mammalian left ventricle was examined in a variety of mammals. The hearts were arrested in situ in animals with an intact thorax by means of an isotonic K+ solution perfused via the aorta and coronaries. The hearts were then fixed by formalin perfusion through the same vessels and the hearts embedded in gelatin. Serial sections were prepared perpendicular to the Apex-Valve axis. On close examination, the muscle fibers show the change in orientation from endocardium to epicardium previously described by others. In addition, the clefts and voids of the inner one-third to inner one-half of the left ventricular wall add another dimension to the fiber direction: the fiber bundles appear to take a curving course from the middle of the wall to the endocardial surface. This pattern was visible in all studied hearts. Speculations are made on the significance of this anatomic arrangement.

Ontogeny of monoamine neurons in the locus coeruleus, raphe nuclei and substantia nigra of the rat. I. Cell differentiation

AbstractThe onset of cell differentiation in the locus coeruleus, dorsal and medial raphe nuclei, and substantia nigra (zona compacta) was studied using the technique of long‐survival H3‐thymidine autoradiography to date neurogenesis. Pregnant female rats were injected with isotope on day 10, 11, 12, 13, 14, 15, 16 or 17 of gestation. Litters were born on day 22 and allowed to survive for 30 days. Brains were prepared for the fluorescence histochemical demonstration of monoamines or fixed by formalin perfusion. Autoradiography was carried out on sections adjacent to those in which monoamine‐nuclear groups were identified by the fluorescence method and also on sections from perfused brains, which were used to facilitate cell counting. The norepinephrine cells of the locus coeruleus began to differentiate (presence of heavily labelled cells) on days 10–13 of gestation, with a peak of heavy labelling on day 12, whereas the 5‐HT cells of the raphe nuclei and the dopamine cells of the substantia nigra began differentiating on days 11–15. In the dorsal raphe nucleus, the peak of heavy labelling occurred on day 14, whereas in the medial nucleus this took place on days 13–14. The substantia nigra, on the other hand, peaked on day 13.Cell differentiation was also studied in the cerebellar Purkinje cells and hippocampal macroneurons (pyramidal and polymorph cells) of areas CA3 and CA4, to which the locus coeruleus has been reported to project. Differentiation of these cells commenced on days 14–15 (Purkinje cells) and 13–18 (hippocampal cells), with both cell types peaking on day 15, a full three days after the peak of heavy labelling in the locus coeruleus.Evidence is discussed for the possible neurotrophic role of monoamine neurons in the neurogenesis of monoaminergic receptive cells.

Single cell isotope injection technique, a tool for studying axonal and dendritic transport.

Radioactive precursors (amino acids and choline) were applied to cat spinal motoneurons by means of micro pipette electrophoresis. The amount injected was determined by the iontophoresis current used. Simultaneous recording of the electrical properties of the nerve cells allowed their identification and the control of the site and effect of injection. At different times after injection (4 min to 3 days) the cats were sacrified by formalin perfusion and autoradiographs were prepared from serial sections of the paraplast embedded spinal cord. This technique provides a high concentration of strictly intracellular radioactivity which is advantageous for the study of intracellular transport. The results indicate that proteins most probably synthetized within the nerve cell soma are transported within the dendrites nearly up to their terminals at a rate of at least 3 mm/h and within the axon at different rates of 0.5 and 1.7 mm/h. Following antidromic stimulation the synthesis of proteins within the nerve cell soma and their export into the axon was increased. Choline derivatives thought to be mainly phospholipids were also transported into dendrites and axon.

実験動物における日本脳炎

Pathology of the experimental Japanese encephalitis (JE) has been investigated using various laboratory animals. In this report, only the pathological examination of rhesus or cynomolgus monkeys weighing 2 to 3kg. is summerized. Used virus strains of JE were Nakayama-NIH and JaTH160 and 0.1 to 0.2ml. or 0.5ml. of virus suspension containing 102.6 to 106.9 BLU (50% suckling mouse lethal unit) were injected intracerebrally or subcutaneously. After inoculation, the symptom, body temperature, and body weight of the infected monkeys were checked every day, and the dead or sacrificed animals in agonal stage were bled for serological examination. Finally autopsy was performed after 10% formalin perfusion. The specimens from each area of the central nervous system (CNS) were frozen by dry ice aceton for fluorescent antibody technique (FAT) assay and virus titration.The peripheral injection with 106 BLU of JaTH160 to 6 monkeys showed inappearent infection. However, the pathological findings regard as the visceral phase previously described by others were not clear, but swelling of the germinal center in the lymphoid tissue was observed, though it could not been decided whether it was the specific change induced by virus infection or not. On the other hand, the intracerebral inoculation revealed the typical encephalitis symptom, and exhibited histologically focal character of the lesion and distribution of foci similar to other animal infection. Neuropathologically, marked changes were observed in the spinal cord, especially, in the anterior horn as if they are observed in Heine-Medin's disease. Therefore, so long as the histopathological changes of the spinal cord is concerned, differential diagnosis between JE and poliomyelitis appeared very difficult. The distribution of foci was observed along the ventricular system as if there is a correlation between the histologic changes of the spinal cord and flow of cerebrospinal fluid (CSF).On the FAT assay, the distribution of viral antigen was exclusively observed in the nerve cell, and no viral antigen was found in the other tissues and cells. These results corresponded with histopathological appearance, but it was infered as FAT findings was rather antecedent to histological appearance.The virus content in each area of CNS was investigated with suckling mice. In early stage of the inoculation, the brain stem centered in the middle brain showed high viral titer. With progressing of the symptom, the spinal cord, especially the lumbal cord, showed the highest titer. These findings coincided with histological appearance. However, the cerebellum showed the high infective titer in spite of its scanty histological change, and CSF and the serum showed so low titer being against one's expectation.The minimal infectious dose of JE virus to monkey was assumed as 102 BLU, although the intensity of pathological changes were not pararell with viral dosis.

A method for the electron microscopic demonstration of cytochrome oxidase in fresh and formalin-prefixed tissues.

Burstone's reaction is adapted for electron microscope purposes. Sections of 300–500 μ thickness cut by free hand are incubated for sixty minutes in N-phenyl-p-phenylenediamine at room temperature. No coupling agent is used. The dye polymere gained is postchelated with 0.5% copper sulphate dissolved in 4% buffered formalin solution. After thorough washing a second postchelation is performed with potassium ferrocyanide. Double postchelation reduces the otherwise high lipid solubility of the reaction product, so that it withstands embedding into epoxy resins. Postfixation with osmic acid adds further contrast to the reaction product. Cell structure — even in central nervous tissue — could be preserved satisfactorily without major loss of enzyme activity by the use of a brief formalin perfusion prior to incubation. The reaction product appears in the form of droplets localized in the mitochondria.

Formalin perfusion for correlative light- and electron-microscopical studies of the nervous system

ABSTRACT The formalin perfusion technique of Pease has been shown to be a satisfactory fixation method for both light and electron microscopy of the nervous system of rat and goldfish, so allowing correlative studies to be made on tissue from the same region of one brain. Immediate post-osmication is not necessary for good ultrastructural preservation and even if it is delayed for 1 week, neuronal structure is adequate for all but detailed cytological work. Unosmicated tissue stained with uranyl acetate and lead citrate showed protein-like structures but not lipids, with membranes appearing as unstained bands. ‘Dark’ glial cells occurred near the surface of the cortex and could not be eliminated by prolonged fixation in situ. ‘ Dark’ neurons were found rarely. It is suggested that, with this method of fixation, any poor preservation of tissue in direct light- and electron-microscopical correlative studies is due to the subsequent processing rather than the initial fixation. Preliminary results on material stained by a new Golgi-Kopsch modification and by reduced silver methods and subsequently examined with the electron microscope suggest that such damage is not extensive.

Some New Electron Microscopical Contributions to the Biology of Neuroglia: Introduction

Publisher Summary This chapter reviews the submicroscopic morphology and function of glial cells published in the “International Review of Neurobiology.” Some recent contributions are related to the problem of fixation of the CNS. A new method of formalin perfusion that gives the best preservation of glial and nerve elements at the electron microscope level is presented in the chapter. For the development of this technique, a study was made on the water changes of the tissue after fixation and the components of the solutions for washing of the blood and fixation were so adjusted as to leave the water content of the brain unchanged. The effect of the perfusion with the washing solution was followed by EEG recordings. Excellent preservation of the glial cells and nerve elements in gray and white matter was observed. This method has permitted new observations on the disposition of the glial membranes. The advantage of this method resides in the use of a cheap, innocuous fixative, and a very simple modus operandi . The total preservation obtained permits a systematic study of any neuro-anatomical region and the carrying out of histophysiological or neuropathological experiments in the CNS. The chapter also discusses the problem of the extracellular space in the CNS and the discrepancy between physiological methods and electron microscopy.