Use of a new mouse schwannoma tumour model to monitor changes in peripheral nerve morphology in Merlin null Schwann cells

Aims Our lab is interested in signals that trigger schwannoma tumour formation and we have previously shown that peripheral nerve injury triggers tumour formation in nerves with Schwann cell-specific loss of the Merlin (NF2) tumour suppressor. The Ras/Raf/MAPK/ERK pathway activity in myelinating Schwann cells is involved in nerve regeneration, causing demyelination and recruitment of inflammatory cells in areas of nerve damage, as well as dedifferentiation of myelinating Schwann cells into a repair-competent state. We have used a mouse model expressing a tamoxifen-inducible Raf-Kinase estrogen receptor fusion protein (Raf-TR) in myelinating Schwann cells of the PNS in either a control wild-type Merlin or Merlin-null background. This allows us to determine the effects of an injury-like signal in Schwann cells and its role in generating schwannoma tumour development. We present here a detailed analysis of the proliferation of Schwann cells within the nerve and morphological changes in PNS structure following Raf-TR activation. Method The P0-promotor driving the Raf-TR transgene is active in myelinating Schwann cells but inactive in the non-myelinating population, allowing specific targeting of the myelinating Schwann cell population. In addition to the Raf-TR gene, the mice exhibit a separate P0-promotor controlled Cre floxed NF2 gene which undergoes Cre-mediated recombinase at embryonic day 13.5 causing NF2 knockout in all developing Schwann cells. Mice aged between 4-6 weeks received intraperitoneal injections of either 2mg Tamoxifen or oil vehicle for 5 consecutive days and were then studied at either 10 or 21 days post-first injection. The peripheral nervous system of the mice was studied with fluorescent immuno-histochemistry staining, semithin sections and transmission electron microscopy (TEM) on sciatic nerves and dorsal root ganglia (DRG). Results Activation of the Ras/Raf/MAPK/ERK pathway in NF2 null Schwann cells led to higher rates of proliferation within sciatic nerves at 10d post-tamoxifen injections. At both 10d and 21d Raf-TR+ NF2-null mice sciatic nerve fascicles were visibly larger with significantly more cell bodies present than controls, however at 21d the rate of proliferation had reduced. In the DRG, proliferation was higher in Raf-TR+ NF2-null mice compared to controls, with proliferation remaining high at 21 days. Quantitative imaging of peripheral nerve semi-thins analysed to date showed no significant difference in the number of myelin rings present in the fascicles between different genotypes. Additionally, dual immuno-histochemistry staining with Myelin Basic Protein and EdU, markers for myelin and proliferation respectively, appeared to show proliferation in the non-myelinating Schwann cell population. Results from staining with other cell markers will also be presented, as well as a detailed analysis of nerve structure using TEM. Conclusion While developmental myelination of Merlin-null Schwann cells appears largely normal, the reaction of Merlin-null Schwann cells in the nerve to an injury signal (activation of the Raf-TR) is remarkably different from those of control nerves. The high levels of proliferation in Merlin-null Schwann cells may be indicative of a higher tumorigenesis potential. While the proliferation of Merlin-null cells does reduce over time in the sciatic nerve, further experiments are now testing whether there may be ongoing tumour growth at other locations in the nervous system that are associated with NF2 tumours in human patients.

Cytoprotective Effect and Clinical Outcome of Perioperative Progesterone in Brain Tumors. A Novel Randomized Mono-Centeric Microscopic Evidence Based Research.

Objectives.The primary end-point of the current study was to to provide contemporary estimate about both cytoplasmic and nuclear effects of intramuscular progesterone therapy prior to craniotomy. Secondary end-points were to track post-operative course and short term (3 months) neurological sequele.Measurments.Cellular neuronal microscopic examination by immuno-histochemistry (Progesterone receptor density ) and by H&E. Allred score for nuclear staining for PR receptors . Post-operative course included time to wean from mechanical ventilation (hours), Length of ICU stay (days) and brain neuro-imaging by brain CT). Short term outcome included sensory, motor and autonomic assessment. Patients.Two hundred fifty two (252) adult patients of both sexes aged 18–60 years, American society of anesthesia (ASA class І-ІІ ) candidate for intra-parenchymal intra-axial elective brain tumors resection , Excluded candidates included refusal to sign document for intervention, urgent craniotomy, history of endometrial or ovarian neoplasia, redo craniotomy, stented coronary arteries or history of deep venous thrombosis. Design. This research was a randomized, double-blind, placebo controlled single center study. Intervention. Two groups, the control group and Progesterone (PR) group. PR group received 1mg/ kg intramuscular progesterone five days before and five days after craniotomy, while Control group received intramuscular isotonic saline daily, five days before and five days after surgery.Setting.Fixtures performed in Minia university hospital, Neurosurgery operative theatre, Floor two (non trauma floor). The study was registered in local ethical committee on April 2020. First patient enrollment was on first of June 2020 and the patient allocation in the rank ended on 30 of December 2020. Main resultsProgesterone therapy successfully increased expression of active nuclear PR receptors on oligodendrocytes and astrocytes, provided cytoplasmic and nuclear neuro-quiescence. Progesterone gave better neurological outcome on 3 month follow- up.Conclusions.Neuronal biopsy examination represented a leap in confirming anti-neuro-inflammatory action offered by progesterone.Name of the registry. Clinical trial .gov.Trial registration number. NCT04414020.Date of registration. 30 of May 2020.URL of trial registry record. ttps://register.clinicaltrials.gov/prs/app/action/SelectProtocol?sid=S0009WLW&selectaction=Edit&uid=U0004XC5&ts=2&cx=-vrqng9

Early Changes in Skeletal Muscle of Young C22 Mice, A Model of Charcot-Marie-Tooth 1A

Background: The C22 mouse is a Charcot-Marie-Tooth 1A transgenic model with minimal axonal loss. Objective: To analyse early skeletal muscle changes resulting from this dysmyelinating neuropathy. Methods: Histology of tibialis anterior muscles of C22 mice and wild type litter mate controls for morphometric analysis and (immuno-)histochemistry for known denervation markers and candidate proteins identified by representational difference analysis (RDA) based on mRNA from the same muscles; quantitative PCR and Western blotting for confirmation of RDA findings. Results: At age 10 days, morphometry was not different between groups, while at 21 days, C22 showed significantly more small diameter fibres, indicating the onset of atrophy at an age when weakness becomes detectable. Neither (immuno-)histochemistry nor RDA detected extrajunctional expression of acetylcholine receptors by age 10 and 21 days, respectively. RDA identified some mRNA up-regulated in C22 muscles, among them at 10 days, prior to detectable weakness or atrophy, integral membrane protein 2a (Itm2a), eukaryotic initiation factor 2, subunit 2 (Eif2s2) and cytoplasmic phosphatidylinositol transfer protein 1 (Pitpnc1). However, qPCR failed to measure significant differences. In contrast, Itm2a and Eif2s2 mRNA were significantly down-regulated comparing 21 versus 10 days of age in both groups, C22 and controls. Western blotting confirmed significant down-regulation of ITM2A protein in C22 only. Conclusion: Denervation-like changes in this model develop slowly with onset of atrophy and weakness at about three weeks of age, before detection of extrajunctional acetylcholine receptors. Altered Itm2a expression seems to being early as an increase, but becomes distinct as a decrease later.

Glucagon Like Peptide-1 and Its Receptor in Human Brain: Distribution of Expression, Functional Implications, Age Related Changes & Species Specific Characteristics

Emerging evidence has shown that the Glucagon like peptide-1 (GLP-1) agonist can be used for treating Alzheimer’s disease, but knowledge of its neural targets is limited. To understand the neural substrates of GLP-1, we have done whole brain mapping for GLP-1 and its receptor (GLP-1R), in 30 human brains. GLP-1 expression was studied by immuno-histochemistry and confirmed by western blot method. GLP-1R gene expression was studied by RT-PCR. GLP-1 expression was seen in most of the cortical areas (maximum in frontal, prefrontal & parietal cortex), diencephalon and brainstem, but not in cerebellum. Protein expression studies validated these results. Highest expression of GLP-1R was found in the frontal cortex. The orbito-frontal cortex and cerebellum had negligible expression. Hippocampus demonstrated significant presence of GLP-1R but patchy immunoreactivity to GLP-1. GLP-1R presence in most of the human cortical regions and absence in cerebellum is the major deviation from the animal brain. Sites which might be of interest in Alzheimer’s have been identified. GLP-1 demonstrated age related decline in most of the areas after 5thdecade. At 60yrs GLP-1 was not found in any of the cortical areas except in the prefrontal cortex but it was present in the sub-cortical areas. Age related profiling of GLP-1 in various brain areas has been analysed, which can have important bearing on understanding the Alzheimer’s. This study provides detailed description of GLP-1 and GLP-1R locations by complete human brain mapping for the first time and may lead to novel treatment options targeting the GLP-1 receptors.

Evidence For and Against Direct Kidney Infection by SARS-CoV-2 in Patients with COVID-19

Despite evidence of multi-organ tropism of SARS-CoV-2 in patients with COVID-19, direct viral kidney invasion has been difficult to demonstrate. The question of whether SARS-CoV-2 can directly infect the kidney is relevant to the understanding of pathogenesis of acute kidney injury and collapsing glomerulopathy in COVID-19. Methodologies to document SARS-CoV-2 infection that have been used include immunohistochemistry, immunofluorescence, reverse transcriptase polymerase chain reaction (RT-PCR), in situ hybridization and electron microscopy. In our review of studies to date we found that SARS-CoV-2 in the kidney of patients with COVID-19 was detected in 18 of 94 (19%) by immuno-histochemistry, 71 of 144 (49%) by RT-PCR and 11 of 84 (13%) by in situ hybridization. In a smaller number of patients with COVID-19 examined by immunofluorescence, SARS-CoV-2 was detected in 10 of 13 (77%). In total, in kidneys from 102 of 235 patients (43.4%), the presence of SARS-CoV-2 was suggested by at least one of the methods used. Despite these positive findings, caution is needed as many other studies have been negative for SARS-CoV-2 presence and it should be noted that when detected it was only in kidneys obtained at autopsy. There is a clear need for studies from kidney biopsies, including those performed at early stages of the COVID-19 associated kidney disease. Development of tests to detect kidney viral infection in urine samples would be more practical as a non-invasive way to evaluate SARS CoV-2 infection during the evolution of COVID-19-associated kidney disease.

Elastic transformation of histological slices allows precise co-registration with microCT data sets for a refined virtual histology approach

Although X-ray based 3D virtual histology is an emerging tool for the analysis of biological tissue, it falls short in terms of specificity when compared to conventional histology. Thus, the aim was to establish a novel approach that combines 3D information provided by microCT with high specificity that only (immuno-)histochemistry can offer. For this purpose, we developed a software frontend, which utilises an elastic transformation technique to accurately co-register various histological and immunohistochemical stainings with free propagation phase contrast synchrotron radiation microCT. We demonstrate that the precision of the overlay of both imaging modalities is significantly improved by performing our elastic registration workflow, as evidenced by calculation of the displacement index. To illustrate the need for an elastic co-registration approach we examined specimens from a mouse model of breast cancer with injected metal-based nanoparticles. Using the elastic transformation pipeline, we were able to co-localise the nanoparticles to specifically stained cells or tissue structures into their three-dimensional anatomical context. Additionally, we performed a semi-automated tissue structure and cell classification. This workflow provides new insights on histopathological analysis by combining CT specific three-dimensional information with cell/tissue specific information provided by classical histology.

Significance of Carbohydrate Antigen 19-9 as a biomarker in Hepatocellular Carcinoma and Cholangiocarcinoma

Background & Aims: Combined hepatocellular - cholangiocarcinomas (cHCC-CCs) are rare malignancies representing less than 1% of all primary liver cancers. Correct preoperative diagnosis is desirable because the frequency of lymph node metastasis in ICC and cHCC-CC, making lymph node dissection a necessity if curative resection to be attempted. This study aimed to investigate the significance of elevated CA19-9 in suspecting a diagnosis of Intrahepatic Cholangiocarcinoma (ICC) “non-invasively” in patients with typical radiological features of HCC. Methods This cross-sectional study was conducted on 54 patients with typical radiological criteria of HCC and elevated CA19-9 level. And were classified into two groups I included 22 patients (40.74%) who were diagnosed as HCC, group II included 30 patients (55.56%) were diagnosed as ICC, and there were 2 patients (3.7%) were diagnosed as cHCC-CC. Tumor markers (AFP and CA19-9), dynamic study (Triphasic CT or Dynamic MRI) were done for all patients. Target liver biopsy was done for histopathology and immuno-histochemistry using specific monoclonal antibodies against Glypican-3, Hep-par1, CK-7, CK-19 and CK-20 were done. Results: There was a statistically significant difference between HCC and ICC as regard CA19-9 and Alpha-fetoprotein (AFP). CA19-9 and AFP cut-offs were ˃ 58.9 U/mL and ˂ 25.8 ng/mL, respectively favoring the diagnosis of ICC, with very high sensitivity and specificity. CA19-9 level was 176.3 and 156.7 U/mL while AFP level was 460 and 170 ng/mL in cHCC-CC cases, respectively. Conclusion: CA19-9 could be a diagnostic marker of ICC in cases of typical radiological criteria of HCC with elevated CA19-9.

An Unusual Presentation of Malignant Melanoma

Melanoma is one of the aggressive malignancy arising from the melanocyte. Melanoma usually occurs in the mucus membrane and choroid. There are few cases who may present with lymph node, breast and other involvement. However, few may have an atypical origin and rarely the origin may be unknown. Its aggressive presentation, diagnosis and prognosis sometimes is very challenging to the physician. The mainstay of the diagnosis remains the histopathology and immuno-histochemistry (IHC). Varieties of treatment have evolved from surgical excision to systemic chemotherapy and immunotherapy with promising result, the treatment is still challenging. A 30 years old lady presented with lump in her both breasts and generalized lymphadenopathy. Excisional biopsy revealed Diffuse large B cell Non Hodgkin lymphoma (NHL) but IHC was in favour of melanoma. The final diagnosis was cutaneous melanoma of trunk with atypical presentation (generalized lymphadenoapthy and bilateral breast metastasis).

Elastic transformation of histological slices allows precise co-registration with microCT data sets for a refined virtual histology approach

Although x-ray based 3D virtual histology is an emerging tool for the analysis of biological tissue, it falls short in terms of specificity when compared to conventional histology. Thus, the aim was to establish a novel approach that combines 3D information provided by microCT with high specificity that only (immuno-)histochemistry can offer. For this purpose, we developed a software frontend, which utilises an elastic transformation technique to accurately co-register various histological and immunohistochemical stainings with free propagation phase contrast synchrotron radiation microCT. We demonstrate that the precision of the overlay of both imaging modalities is significantly improved by performing our elastic registration workflow, as evidenced by calculation of the displacement index. To illustrate the need for an elastic co-registration approach we examined specimens from a mouse model of breast cancer with injected metal-based nanoparticles. Using the elastic transformation pipeline, we were able to co-localise the nanoparticles to specifically stained cells or tissue structures into their three-dimensional anatomical context. Additionally, we performed a semi-automated tissue structure and cell classification. This workflow provides new insights on histopathological analysis by combining CT specific three-dimensional information with cell/tissue specific information provided by classical histology.

CASE REPORT OF CUTANEOUS MASTOCYTOSIS

Mastocytosis is a heterogeneous group of disorder characterized by abnormal growth and accumulation of mast cells in skin and other organs such as bones, gastrointestinal tract, liver, spleen and lymph node. A 3 months old male child, presented with multiple, discrete, reddish, papulo-nodular lesions all over body which became more prominent and itchy after a warm bath and crying. Darrier sign was positive and systemic examination was normal. SCORMA index was calculated. Skin biopsy was suggestive of Langherans cell histiocytosis. Immuno- histochemistry profile was positive for CD68, Vimentin, LCA and CD117. This condition needs high index of suspicion and should be differentiated from other causes of urticaria. It is a benign condition, but parental counselling and avoidance of trigger factors is essential. It is imperative to rule out malignancy and systemic complications in a case of cutaneous mastocytosis.