Routine Electron Microscopy Research Articles

Cellular mechanisms of the blood-brain barrier opening induced by ultrasound in presence of microbubbles

Local blood-brain barrier (BBB) opening is an advantageous approach for targeted drug delivery to the brain. Recently, it has been shown that focused ultrasound (US) exposures (sonications), when applied in the presence of preformed gas bubbles, caused magnetic-resonance (MR) proven reversible opening of the BBB in targeted locations. The cellular mechanisms of such transient barrier disruption are largely unknown. We investigated US-induced changes in endothelial cell fine morphology that resulted in the BBB opening in rabbits. To obtain evidence for the passage of blood-borne macromolecules through the opened transvascular routes, an immunocytochemical procedure for endogenous immunoglobulinG (IgG) was performed, in addition to the routine electron microscopy. An increased number of vesicles and vacuoles, fenestration and channel formation, as well as opening of some tight junctions, were seen in capillaries after low-power (0.55 W) sonication. Immunosignals presented in some of the vesicles and vacuoles, in the cytoplasmic channels and, so rarely, in intercellular clefts; immunosignals could also be seen in neuropil around the blood vessels. Damage to the cellular ultrastructure was not seen in these areas. However, cell destruction and leakage of IgG through defects of the endothelial lining took place at 3 W sonications. The data reveals that several mechanisms of transcapillary passage are possible after such sonications: 1. transcytosis; 2. endothelial cell cytoplasmic openings—fenestration and channel formation; 3. opening of a part of tight junctions; and 4. free passage through the injured endothelium (with the higher power sonications). These findings could be considered in further development of the strategy for drug delivery to brain parenchyma. (E-mail: nsheikov@bwh.harvard.edu)

Inherited retinal dysplasia and persistent hyperplastic primary vitreous in Miniature Schnauzer dogs

The objectives of this study were to define the clinical syndrome of retinal dysplasia and persistent primary vitreous in Miniature Schnauzer dogs and determine the etiology. We examined 106 Miniature Schnauzers using a biomicroscope and indirect ophthalmoscope. The anterior and posterior segments of affected dogs were photographed. Four enucleated eyes were examined using routine light microscopy and scanning electron microscopy. A pedigree was constructed and related dogs were test-bred to define the mode of inheritance of this syndrome. Congenital retinal dysplasia was confirmed in 24 of 106 related Miniature Schnauzer dogs. Physical and postmortem examinations revealed that congenital abnormalities were limited to the eyes. Biomicroscopic, indirect ophthalmoscopic, and neuro-ophthalmic examinations confirmed that some of these dogs were blind secondary to bilateral retinal dysplasia and detachment (nonattachment) (n = 13), and the remainder had generalized retinal dysplasia (n = 11). Fifteen of these dogs were also diagnosed with unilateral (n = 9) or bilateral (n = 6) persistent hyperplastic primary vitreous. Nutritional, infectious, or toxic etiologies were not evident on physical, postmortem, light microscopic, or transmitting and scanning electron microscopic examination of four affected Miniature Schnauzers. We examined the pedigree and determined that an autosomal recessive mode of inheritance was most likely. Three test-bred litters including those from affected parents, carrier and affected parents, and carrier parents confirmed this mode of inheritance. This study confirms that retinal dysplasia and persistent hyperplastic primary vitreous is a congenital abnormality that is inherited as an autosomal recessive condition in Miniature Schnauzers.

Answers to MCQs

This study was conducted to investigate the nature of colonic metaplasia in ileo-anal pouches and incidence/frequency of pouchitis in the same. Biopsy specimens from 8 patients with functioning ileal pouches were studied using routine histology, mucin histochemistry and electron microscopy, over a 2 – year period. All 8 patients had villous abnormalities in the form of blunting of villi and sub total or partial villous atrophy. 6 patients had an increase in the goblet cell population and Paneth cell hyperplasia. These changes were supported by electron microscopic findings of a decrease in number and flattening of ileal type microvilli and their transformed morphologic resemblance to colonic type microvilli. All the ileal pouches also had acquired colorectal type sulphomucin, when sections stained with Alcian-blue and High Iron Diamine – Alcian blue, were studied. However, no case of pouchitis as defined in literature, was found in this study.

Ultrastructural observations of efferent terminals in the crista Ampullaris of the toadfish, opsanus tau.

The present study was conducted to visualize the ultrastructural features of vestibular efferent boutons in the oyster toadfish, Opsanus tau. The crista ampullaris of the horizontal semicircular canal was processed for and examined by routine transmission electron microscopy. The results demonstrate that such boutons vary in size and shape, and contain a heterogeneous population of lucent vesicles with scattered dense core vesicles. Efferent contacts with hair cells are characterized by local vesicle accumulations in the presynaptic terminal and a subsynaptic cistern in the postsynaptic region of the hair cell. Serial efferent to hair cell to afferent synaptic arrangements are common, particularly in the central portion of the crista. However, direct contacts between efferent terminals and afferent neurites were not observed in our specimens. The existence of serial synaptic contacts, often with a row of vesicles in the efferent boutons lining the efferent-afferent membrane apposition, suggests that the efferent influence on the crista may involve both synaptic and nonsynaptic, secretory mechanisms. Further, it is suggested that differences in more subtle aspects of synaptic architecture and/or transmitter and receptor localization and interaction may render the efferent innervation of the peripheral crista less effective in influencing sensory processing.

Immunohistochemical and ultrastructural markers suggest different origins for cuboidal and polygonal cells in pulmonary sclerosing hemangioma

We report the morphological characteristics of 30 cases of sclerosing hemangioma (SH) of the lung and explore the histological origin of the major cells in these tumors. In addition to routine light and electron microscopy, immunohistochemistry was performed by using 12 monoclonal primary and 5 polyclonal primary antibodies. These included surfactant protein B (SP-B), thyroid transcription factor-1 (TTF-1), mast cell trypsin, CD68, epithelial antigen markers (high molecular weight cytokeratin, low molecular weight cytokeratin [CK-L], epithelial membrane antigen [EMA], cancer embryonic antigen), mesothelial antigen, neuroendocrine markers (neuron-specific enolase [NSE], chromogranin A, synaptophysin, calcitonin, adrenocorticotropic hormone, human growth hormone [hHG]), vimentin, and CD34. Surface cuboidal cells have short microvilli and have lamellar bodies in their cytoplasm. They can sometimes merge into multinuclear giant cells. Immunohistochemical results showed that these cells are strongly positive for SP-B, TTF-1, CK-L, EMA, and cancer embryonic antigen, whereas polygonal cells, previously also described as round or pale cells, were strongly positive for vimentin and TTF-1, and positive or weakly positive for 2 to 3 kinds of neuroendocrine markers. Sparse neuroendocrine granules and abundant microfilaments were observed in their cytoplasm. Some cell clusters in the solid regions were positive for SP-B and EMA. Mast cells existed sparsely in almost every field. Both cuboidal and polygonal cells were negative to CD34 and mesothelial antigen staining. We conclude that cuboidal cells of SH originate from reactive proliferating type II pneumocytes, which can fuse into multinuclear giant cells. Polygonal cells, as true tumor cells, likely originate from multipotential primitive respiratory epithelium and possess the capability for multipotential differentiation. The antibodies of SP-B, TTF-1, vimentin, and CK-L are very helpful to diagnosis and differential diagnosis of SH.

Effect of valproic acid on fetal and maternal organs in the mouse: a morphological study.

Valproic acid (VPA) is an antiepileptic drug used clinically. Because of its known teratogenic properties VPA is not recommended for women of child bearing age. The present study was designed to assess the effects of VPA on both fetal and maternal organs. Randomized groups of pregnant mice were treated as follows: Group 1 (n = 10) 500 mg/kg VPA/day on gestation days 8-11; Group 2 (n = 10) 600 mg/kg VPA/day on gestation days 8-11; and Group 3 (n = 4) saline-injected controls. On gestation day 18, the pregnant mice were euthanized, fetuses collected and prepared for scanning electron microscopy. In addition, fetal and maternal organs were processed for routine histology, immunohistochemistry for growth factors (TGF alpha, beta-1, beta-2 and EGF) and transmission electron microscopy. Scanning microscopy revealed specific lesions induced by VPA in the fetus, namely spina bifida occulta, exencephaly, and exophthalmia. On the other hand, there were no detectable morphological changes in fetal or maternal organs by routine histology, immunohistochemistry or electron microscopy. The data suggest that the lesions present in the fetus are due to a direct effect by VPA on retinoic acid, a ubiquitous compound that has a role in normal development, rather than the lack of transport of sufficient nutrients to the fetus as a result of placental insufficiency due to VPA-induced toxicity.

Increase of melanogenesis by retinoic acid: an ultrastructural and morphometric study

Melanin is a dark pigment protecting the skin against UV radiation in some organisms. Studies on invasion and metastasis using retinoic acid as inhibitor agent are well known, but its role in melanin production (melanogenesis), especially at ultrastructural level and using morphometry were not well studied. In the present study, we analyzed the effects of retinoic acid on the melanosomes in B16F10 melanoma cells. These organelles were identified and quantified using routine electron microscopy and the specific HMB45 antibody. Other approaches such as immunofluorescence, and flow cytometry were also used. Our results indicated that retinoic acid increased the melanogenesis process in B16F10 melanoma cells. Furthermore, this work also provided evidence that this substance interferes at the subcellular level altering the numerical density of melanosomes, as well as the relative volume of the nucleus and nucleolus. In addition, the cells displayed altered morphology and an increase in the percentage of the relative volume of melanosomes, mainly the stages II–III and IV, leading to melanin formation. Furthermore, a decrease in the cells number after retinoic acid treatment was also observed.

The high incidence of BK polyoma virus infection among renal transplant recipients in India.

BK polyoma virus causes allograft dysfunction as a result of tubulo-interstitial nephritis in 2% to 5% of renal transplant recipients. The incidence of BK virus infection among renal transplant recipients in India is unknown. We used routine histologic examination, immunohistochemistry, and electron microscopy to retrospectively screen for BK polyoma virus in 414 renal allograft biopsy specimens from 321 transplant recipients presenting with allograft dysfunction. All patients had received a combination of cyclosporine, azathioprine, and prednisolone. A total of 30 biopsy specimens (9.3%) were positive for BK polyoma virus, suggesting a high incidence of this infection in Indian transplant recipients. BK virus infection coexisted with acute rejection in a majority of patients. This is the first report of this infection among Indian renal transplant recipients.

Cytological and immunocytochemical characterization of the insulin secreting insulinoma cell line RINm5F.

The rat insulinoma cell line RINm5F, an insulin secreting pancreatic beta cell line, has been used as an attractive model for basic studies of the mechanisms of insulin secretion and, more recently, as a model for the development of alternative methods for the treatment of diabetes. To elucidate the cytological properties and expression patterns of hormones of the gastro-entero-pancreatic system, suspensions of RINm5F cells were investigated by various methods including immunocytochemistry on serial semithin sections, quantitative immunocytochemistry, routine electron microscopy, immuno-electron microscopy, in situ hybridization, and TUNEL technique. At the ultrastructural level, several phenotypes of RIm5F cells were characterized by differences in the number, shape, size, and density of their secretory granules. The most common type contained a mixture of round granules varying in size and electron density. A second type predominantly contained relatively large, moderately dense granules. Moreover, a minority of cells was characterized by the occurrence of polymorphous electron dense granules or the complete absence of any secretory granules. The immunohistochemical data showed that, among the established islet hormones, insulin was present in more than 50% of cells, whereas glucagon and somatostatin occurred only sporadically. Though cells positive for pancreatic polypeptide (PP) were not found, PP-related peptides (NPY and PYY) however could be detected in a minority of cells. The great majority of RINm5F cells were immunoreactive for chromogranin B (CgB), followed by insulin, chromogranin A (CgA), and serotonin (5-HT). In addition to intercellular differences in the density of immunostaining, numerous colocalizations of immunoreactivities were found, suggesting that RINm5F cells represent a mixture of subtypes concerning the individual pattern of hormone expression. The present results reveal a wide range of heterogeneity with respect to the morphology and especially the hormone content between individual RINm5F cells.

Immunophenotypic characterization and depletion of pulmonary intravascular macrophages of horses.

Pulmonary intravascular macrophages (PIMs) are present in horses and are believed to increase their sensitivity to endotoxin-induced cardio-pulmonary shock. However, owing to a lack of a marker for PIMs and the inability to isolate them, their precise contributions in the horse remain unknown. We designed this study to identify an immuno-phenotypic marker for PIMs and to develop a protocol for their transient depletion with gadolinium chloride (GC). GC is a lanthanide that has been used to deplete liver and lung macrophages. The horses (N = 15) were divided into control (n = 5) and GC-treated (n = 10) groups and the lung samples were examined by routine and immunocytochemical light and electron microscopy. GC-treated horses were euthanized at 48 h (n = 6) and 72 h (n = 4) post-treatment. The PIMs reacted with MAC-387 but not with ED-1 and CD-68 anti-macrophage antibodies. GC reduced the number of PIMs in horses at 48 and 72 h compared with the control (p < 0.05). There were increased intravascular TUNEL-positive cells in GC-treated horses and electron microscopy showed apoptotic PIMs in these horses. These data show that MAC-387 is a reliable marker for PIMs and GC is a safe tool to reduce the number of PIMs.

Synaptic lesions and synaptophysin distribution change in spinal motoneurons at early stages following sciatic nerve transection in neonatal rats

The purpose of this investigation was to evaluate the rate of synaptic stripping, changes in the synaptophysin distribution, and synapses ultrastructure of spinal motoneurons at early stages of sciatic nerve axotomy in newborn rats. Seven groups were used in the experiment, which were sacrificed after 1, 3, 6, 12, 24, 48 and 72 h. L4–L6 spinal segments from the animals of the above groups were prepared and processed for indirect immunoperoxidase. Accordingly, tissue samples were prepared from similar groups for routine electron microscopy. Synaptophysin-labeled motoneurons were classified into intact, partial, cytoplasmic and negative patterns. Synaptophysin immunoreactivity in both the axotomized and non-axotomized sides showed no negative pattern at the first three time points, while this pattern significantly increased in the 12, 24, 48 and 72 h groups at the axotomized side, moreover, there was a progressive reduction in the percentage of the intact pattern at the axotomized side. The percentages of the cytoplasmic and partial patterns also increased during the time course. The rate of synaptic stripping was five time higher in the axotomized side than that of non-axotomized side. The results of the ultrastructural study showed synaptic membranes irregularity, synaptic vesicles displacement, synaptic membrane detachment and ensheathment of degenerated synapses. The conclusion of the study was that early synaptophysin immunoreactivity changes were seen in both the axotomized and non-axotomized sides, but the rate of change in the axotomized side was more rapid than that of the non-axotomized side.

Immunohistochemical demonstration of the small GTPase RhoA on epoxy-resin embedded sections

The purpose of the present study was to establish a method for light microscopical immunohistochemical localization of the small G protein RhoA on specimens treated and embedded for routine transmission electron microscopy. There are advantages in antigen immunolocalization on resin semi-thin sections compared to cryostat or paraffin sections: the preservation of morphological details, the well-defined immunoprecipitate localization and the possibility to correlate the immunohistochemical results with those obtained by electron microscope on neighbouring sections. These advantages are particularly useful for the subcellular localization of low molecular weight proteins such as RhoA, a small G protein able to cycle from the inactive cytoplasmic form to the plasma membrane-bound active form.

Adhesive interactions between CD34(+)-derived dendritic cell precursors and dermal microvascular endothelial cells studied by scanning electron microscopy.

Dendritic cells are migratory cells. Before they extravasate from the circulation into the skin across capillary blood vessel walls, they have to interact with endothelial cells. Using a fluorimetric adhesion assay, we have recently shown that CD34(+)-derived dendritic cell precursors are able to bind to resting and stimulated dermal microvascular endothelial cells. In the present study, we attempted to visualize this process at an ultrastructural level. CD34(+) progenitor cells were purified from human cord blood samples by means of immunomagnetic beads, and dendritic cells were generated by culture in the presence of GM-CSF, TNF-alpha and hSCF for 5 days. Immature CD83(-) CD86(low) dendritic cells were added to human dermal microvascular endothelial cells grown to confluence on membrane chambers. After 2 h, unbound dendritic cell precursors were removed, and bound cells were prepared for routine scanning electron microscopy. We found that (1). dendritic cell precursors firmly adhere to microvascular endothelial cells, enveloping them with their surface processes; (2). dendritic cell precursors are extremely deformable as they squeeze through the dense network of microvascular endothelial cells; (3). microvascular endothelial cells form, in part, a multi-layered network rather than the typical cobblestone pattern as seen by phase-contrast microscopy. The morphology of dendritic cell precursors and of human dermal microvascular endothelial cells was examined here, for the first time, by scanning electron microscopy. These data further emphasize that CD34(+)-derived dendritic cells efficiently adhere to dermal microvascular endothelial cells.

Radiological quiz

This study was conducted to investigate the nature of colonic metaplasia in ileo-anal pouches and incidence/frequency of pouchitis in the same. Biopsy specimens from 8 patients with functioning ileal pouches were studied using routine histology, mucin histochemistry and electron microscopy, over a 2 – year period. All 8 patients had villous abnormalities in the form of blunting of villi and sub total or partial villous atrophy. 6 patients had an increase in the goblet cell population and Paneth cell hyperplasia. These changes were supported by electron microscopic findings of a decrease in number and flattening of ileal type microvilli and their transformed morphologic resemblance to colonic type microvilli. All the ileal pouches also had acquired colorectal type sulphomucin, when sections stained with Alcian-blue and High Iron Diamine – Alcian blue, were studied. However, no case of pouchitis as defined in literature, was found in this study.

Restorative proctocolectomy with ileo-anal reservoir, a histopathological, histochemical, and electron microscopic study

This study was conducted to investigate the nature of colonic metaplasia in ileo-anal pouches and incidence / frequency of pouchitis in the same. Biopsy specimens from 8 patients with functioning ileal pouches were studied using routine histology, mucin histochemistry and electron microscopy, over a 2 - year period. All 8 patients had villous abnormalities in the form of blunting of villi and sub total or partial villous atrophy. 6 patients had an increase in the goblet cell population and Paneth cell hyperplasia. These changes were supported by electron microscopic findings of a decrease in number and flattening of ileal type microvilli and their transformed morphologic resemblance to colonic type microvilli. All the ileal pouches also had acquired colorectal type sulphomucin, when sections stained with Alcian-blue and High Iron Diamine - Alcian blue, were studied. However, no case of pouchitis as defined in literature, was found in this study.

Tryptophan deficiency arrests chromatin breakdown in secondary lens fibers of rats

Tryptophan deficiency is known for long time to cause cataract in rats. However, up till now the underlying mechanism is still enigmatic. Histological studies showed an extended lens bow suggesting that the normal breakdown of nuclei in the lens fibres is arrested under these conditions. Using advanced ultrastructural techniques we aimed to clarify this aberrant final differentiation of lens fibres. Albino and pigmented rats were permanently or intermittently raised on a tryptophan deficient diet for 12 and 16 weeks, respectively. Rats of the same age raised on a normal diet served as controls. Lenses were treated for light and electron microscopy. For histology sections were stained for DNA and γ-crystallins. In addition to routine transmission electron microscopy (TEM), ultrathin sections were subjected to electron tomography and energy dispersive X-ray microanalysis (EDX). Histology verified the extended lens bow for albino and pigmented rats and showed that in the intermittent period of normal diet the fibre nuclei are broken down as in controls. It was further shown that γ-crystallins are co-localized with DNA in the nuclear domain. TEM revealed that during final differentiation nuclear chromatin becomes highly compacted in a chromosome-like manner and than rapidly evanesces in control rats. This compacted stage persists indefinitely in the tryptophan deficient rats. Electron tomography showed that during differentiation chromatin is first uncoiled to 30 nm solenoids, subsequently to highly compacted 10 nm beads-on-a-string fibrils and than is segregated from the nuclear proteins. EDX revealed that the late stage persisting nuclei consist of domains rich in DNA associated with histones and in domains with mainly proteins. This study corroborates previous findings on the final breakdown of nuclei of lens fibres. It further shows that the chromatin is ultimately uncoiled to beads-on-a-string fibrils and that as the last step chromatin is broken down at this unmasked stage. Except for this last step nuclear breakdown is identical in control and tryptophan deficient rats suggesting that it is not the availability of tryptophan for protein synthesis in general which causes the arrest. Two alternatives for this final arrest are discussed. A low tryptophan content, most pronounced in deeper cortical layers, may inhibit the late synthesis of the DNases and proteases necessary for chromatin breakdown. The radical scavenging by indoleamine 2,3-dioxygenase, which cleaves the pyrrole ring of tryptophan to form formylkynurenine using free oxygen radicals, is impaired by low levels of tryptophan. This decreased scavenging of oxygen radicals will expose the catalytic enzymes for chromatin breakdown, residing in the nucleus in an inactive form for quite a long period, to high levels of oxygen radicals and may affect the activity of these enzymes and therefore the execution of the chromatin breakdown.

Ultrastructural immunohistochemical localization of gastrin, somatostatin and serotonin in endocrine cells of human antral gastric mucosa

Five types of endocrine cells are found in the human antral gastric mucosa: gastrin (G) cells, somatostatin (D) cells, enterochromaffin (EC) cells and cells with an unknown secretory product (D1 cells and P cells). The content of secretory granules, gastrin, somatostatin and serotonin, was evaluated using electron microscopic immunohistochemistry and was compared with the granular content in G cells, D cells and EC cells as determined by routine electron microscopy. Semi-quantitative scoring of the granular content was performed on a scale 1-4 (empty-full). The content of gastrin (2.5 +/- 0.2) and somatostatin (3.3 +/- 0.2) in the granules was not different from the granular content in G cells (2.5 +/- 0.3; p > 0.05) and D cells (3.5 +/- 0.2; p > 0.05). Gastrin was also found in G cells in a nongranular form. The content of serotonin in granules (2.8 +/- 0.3) was smaller than the granular content in EC cells (3.7 +/- 0.2; p < 0.05). In intermediate-full and intermediate-empty granules, serotonin was localized in the periphery of granules whereas the granular content in EC cells was localized in an eccentric or central pattern. The granular content of D1 cells and P cells was 3.8 +/- 0.2, and 3.4 +/- 0.2, respectively. It is concluded that gastrin and somatostatin immunostaining in granules of G cells and D cells reflects the granular content in G cells and D cells, respectively, whereas serotonin immunostaining does not agree with the granular content of EC cells.

Staurosporine-induced apoptosis and hydrogen peroxide-induced necrosis in two human breast cell lines.

The use of apoptosis-inducing agents in the treatment of malignant cancer is increasingly being considered as a therapeutic approach. In this study, the induction of apoptosis and necrosis was examined in terms of temporal dose responses, comparing a malignant and nonmalignant breast cell line. Staurosporine (SSP)-induced apoptosis and H2O2-induced necrosis were evaluated by two cytotoxicity assays, neutral red (NR) and methyl-thiazolyl tertrazolium (MTT), in comparison with a differential dye uptake assay, using Hoechst33342/propidium iodide (Hoechst/PI). Confirmatory morphological assessment was also performed by routine resin histology and transmission electron microscopy. Cell viability was assessed over a 0.5–48 h time course. In nonmalignant HBL-100 cells, 50 nM SSP induced 100% apoptosis after a 48 h exposure, while the same exposure to SSP caused only 4% apoptosis in metastatic T47D cells. Although complete apoptosis of both cell lines was induced by 50 μM SSP, this effect was delayed in T47D (24 h) compared with HBL-100 (4 h). Results also showed that neither MTT or NR can distinguish between the modes of cell death, nor detect the early onset of apoptosis revealed by Hoechst/PI.

Nipah Virus Infection: Pathology and Pathogenesis of an Emerging Paramyxoviral Zoonosis

In 1998, an outbreak of acute encephalitis with high mortality rates among pig handlers in Malaysia led to the discovery of a novel paramyxovirus named Nipah virus. A multidisciplinary investigation that included epidemiology, microbiology, molecular biology, and pathology was pivotal in the discovery of this new human infection. Clinical and autopsy findings were derived from a series of 32 fatal human cases of Nipah virus infection. Diagnosis was established in all cases by a combination of immunohistochemistry (IHC) and serology. Routine histological stains, IHC, and electron microscopy were used to examine autopsy tissues. The main histopathological findings included a systemic vasculitis with extensive thrombosis and parenchymal necrosis, particularly in the central nervous system. Endothelial cell damage, necrosis, and syncytial giant cell formation were seen in affected vessels. Characteristic viral inclusions were seen by light and electron microscopy. IHC analysis showed widespread presence of Nipah virus antigens in endothelial and smooth muscle cells of blood vessels. Abundant viral antigens were also seen in various parenchymal cells, particularly in neurons. Infection of endothelial cells and neurons as well as vasculitis and thrombosis seem to be critical to the pathogenesis of this new human disease.

The effects of combined fluid percussion traumatic brain injury and unilateral entorhinal deafferentation on the juvenile rat brain

The current study was designed to address the effects of traumatic brain injury (TBI) on plasticity and reorganization in the juvenile brain. Given that two of the major pathological sequelae of TBI involve a generalized neuroexcitation insult and diffuse axonal injury, we have employed models of these pathologies, delivered either independently or in combination, to examine their effects on injury-induced synaptic reorganization of the dentate gyrus in the developing rat. Postnatal day 28 rats received either sham, central fluid percussion traumatic brain injury (TBI), unilateral entorhinal cortical lesion (UEC), or TBI+UEC (TUEC) injury. Cognitive performance was assessed in the Morris water maze (MWM) between 11 and 15 days post-injury and the brains were processed for synaptophysin immunohistochemistry and routine electron microscopy. The MWM results revealed that TBI or UEC lesions delivered independently do not produce significant morbidity in P28 rats. However, when these injuries are combined, they reveal significant deficits in the MWM, accompanied by measurable changes in the distribution of presynaptic synaptophysin immunoreactivity over the deafferented dentate molecular layer. These observations are further supported by qualitative ultrastructural alterations in synaptic architecture in the same subregions of the dentate neuropil. The present findings show that the resilience of the immature brain following TBI is reduced when neuroexcitatory insult is combined with deafferentation. Moreover, when deafferented tissue is assessed morphologically, evidence exists for aberrant plasticity and abnormal synaptic reorganization in the juvenile brain.