Transcriptomics of acute myeloid leukaemia core bone marrow biopsies reveals distinct therapy response-specific osteo-mesenchymal profiles.

While the bone marrow (BM) microenvironment is significantly remodelled in acute myeloid leukaemia (AML), molecular insight into AML-specific alterations in the microenvironment has been historically limited by the analysis of liquid marrow aspirates rather than core biopsies that contain solid-phase BM stroma. We assessed the effect of anthracycline- and cytarabine-based induction chemotherapy on both haematopoietic and non-haematopoietic cells directly in core BM biopsies using RNA-seq and histological analysis. We compared matched human core BM biopsies at diagnosis and 2 weeks after cytarabine- and anthracycline-based induction therapy in responders (<5% blasts present after treatment) and non-responders (≥5% blasts present after treatment). Our data indicated enrichment in vimentin (VIM), platelet-derived growth factor receptor beta (PDGFRB) and Snail family transcriptional repressor 2 (SNAI2) transcripts in responders, consistent with the reactivation of the mesenchymal population in the BM stroma. Enrichment of osteoblast maturation-related transcripts of biglycan (BGN), osteopontin (SPP1) and osteonectin (SPARC) was observed in non-responders. To the best of our knowledge, this is the first report demonstrating distinct osteogenic and mesenchymal transcriptome profiles specific to AML response to induction chemotherapy assessed directly in core BM biopsies. Detailing treatment response-specific alterations in the BM stroma may inform optimised therapeutic strategies for AML.

A pattern-based approach to the interpretation of skeletal muscle biopsies.

The interpretation of muscle biopsies is complex and provides the most useful information when integrated with the clinical presentation of the patient. These biopsies are performed for workup of a wide range of diseases including dystrophies, metabolic diseases, and inflammatory processes. Recent insights have led to changes in the classification of inflammatory myopathies and have changed the role that muscle biopsies have in the workup of inherited diseases. These changes will be reviewed. This review follows a morphology-driven approach by discussing diseases of skeletal muscle based on a few basic patterns that include cases with (1) active myopathic damage and inflammation, (2) active myopathic damage without associated inflammation, (3) chronic myopathic changes, (4) myopathies with distinctive inclusions or vacuoles, (5) biopsies mainly showing atrophic changes, and (6) biopsies that appear normal on routine preparations. Each of these categories goes along with certain diagnostic considerations and pitfalls. Individual biopsy features are only rarely pathognomonic. Establishing a firm diagnosis therefore typically requires integration of all of the biopsy findings and relevant clinical information. With this approach, a muscle biopsy can often provide helpful information in the diagnostic workup of patients presenting with neuromuscular problems.

BRAF-mutated suprasellar glioblastoma mimicking craniopharyngioma: illustrative case.

BACKGROUND: Suprasellar masses commonly include craniopharyngiomas and pituitary adenomas. Suprasellar glioblastoma is exceedingly rare with only a few prior case reports in the literature. Suprasellar glioblastoma can mimic craniopharyngioma or other more common suprasellar etiologies preoperatively. OBSERVATIONS: A 65-year-old male with no significant history presented to the emergency department with a subacute decline in mental status. Work-up revealed a large suprasellar mass with extension to the right inferior medial frontal lobe and right lateral ventricle, associated with significant vasogenic edema. The patient underwent an interhemispheric transcallosal approach subtotal resection of the interventricular portion of the mass. Pathological analysis revealed glioblastoma, MGMT partially methylated, with a BRAF V600E mutation. LESSONS: Malignant glioblastomas can mimic benign suprasellar masses and should remain on the differential for a diverse set of brain masses with a broad range of radiological and clinical features. For complex cases accessible from the ventricle where the pituitary complex cannot be confidently preserved via a transsphenoidal approach, an interhemispheric approach is also a practical initial surgical option. In addition to providing diagnostic value, molecular profiling may also reveal therapeutically significant gene alterations such as BRAF mutations.

Stem cell phenotype predicts therapeutic response in glioblastomas with MGMT promoter methylation.

A growing body of evidence supports the presence of a population of cells in glioblastoma (GBM) with a stem cell-like phenotype which shares certain biological markers with adult neural stem cells, including expression of SOX2, CD133 (PROM1), and NES (nestin). This study was designed to determine the relationship between the expression of these stem cell markers and the clinical outcome in GBM patients. We quantified the intensity of expression of the proteins CD133 and SOX2 by immunohistochemistry (IHC) in a cohort of 86 patients with IDH-wildtype GBM, and evaluated patient outcomes using Kaplan-Meier and Cox proportional hazards analysis. In our patients, MGMT promoter methylation status and age were predictors of overall survival and progression free survival. The levels of SOX2 and CD133 were not associated with outcome in univariate analysis; however, stratification of tumors based on low or high levels of CD133 or SOX2 expression revealed that MGMT methylation was a predictor of progression-free survival and overall survival only for tumors with high levels of expression of CD133 or SOX2. Tumors with low levels of expression of CD133 or SOX2 did not show any relationship between MGMT methylation and survival. This relationship between MGMT and stem cell markers was confirmed in a second patient cohort, the TCGA dataset. Our results show that stratification of GBM by the level of expression of CD133 and SOX2 improved the prognostic power of MGMT promoter methylation status, identifying a low-expressing group in which the clinical outcome is not associated with MGMT promoter methylation status, and a high-expressing group in which the outcome was strongly associated with MGMT promoter methylation status. These findings support the concept that the presence of a high stem cell phenotype in GBM, as marked by expression of SOX2 or CD133, may be associated with the clinical response to treatment.

Isolation and characterization of a population of stem-like progenitor cells from an atypical meningioma.

The majority of meningiomas are benign tumors associated with favorable outcomes; however, the less common aggressive variants with unfavorable outcomes often recur and may be due to subpopulations of less-differentiated cells residing within the tumor. These subpopulations of tumor cells have tumor-initiating properties and may be isolated from heterogeneous tumors when sorted or cultured in defined medium. We report the isolation and characterization of a population of tumor-initiating cells derived from an atypical meningioma. We identify a tumor-initiating population from an atypical meningioma, termed meningioma-initiating cells (MICs). These MICs self-renew, differentiate, and can recapitulate the histological characteristics of the parental tumor when transplanted at 1000 cells into the flank regions of athymic nude mice. Immunohistochemistry reveals stem-like protein expression patterns similar to neural stem and progenitor cells (NSPCs) while genomic profiling verified the isolation of cancer cells (with defined meningioma chromosomal aberrations) from the bulk tumor. Microarray and pathway analysis identifies biochemical processes and gene networks related to aberrant cell cycle progression, particularly the loss of heterozygosity of tumor suppressor genes CDKN2A (p16(INK4A)), p14(ARF), and CDKN2B (p15(INK4B)). Flow cytometric analysis revealed the expression of CD44 and activated leukocyte adhesion molecule (ALCAM/CD166); these may prove to be markers able to identify this cell type. The isolation and identification of a tumor-initiating cell population capable of forming meningiomas demonstrates a useful model for understanding meningioma development. This meningioma model may be used to study the cell hierarchy of meningioma tumorogenesis and provide increased understanding of malignant progression.

Pulmonary tumor embolism: A retrospective study over a 30-year period.

BACKGROUND: Pulmonary tumor embolism (PTE) is difficult to detect before death, and it is unclear whether the discrepancy between antemortem clinical and postmortem diagnosis improves with the advance of the diagnostic technologies. In this study we determined the incidence of PTE and analyzed the discrepancy between antemortem clinical and postmortem diagnosis. METHODS: We performed a retrospective autopsy study on patients with the history of malignant solid tumors from 1990 to 2020 and reviewed all the slides of the patients with PTE. We also analyzed the discrepancies between antemortem clinical and postmortem diagnosis in 1999, 2009 and 2019 by using the Goldman criteria. Goldman category major 1 refers to cases in which an autopsy diagnosis was the direct cause of death and was not recognized clinically, but if it had been recognized, it may have changed treatment or prolonged survival. RESULTS: We found 20 (3%) cases with PTE out of the 658 autopsy cases with solid malignancies. Out of these 20 cases, urothelial carcinoma (30%, 6/20) and invasive ductal carcinoma of the breast (4/20, 20%) were the most common primary malignancies. Seven patients with shortness of breath died within 3-17 days (average 8.4±2.2 days) after onset of the symptoms. Pulmonary embolism was clinically suspected in seven out of twenty (35%, 7/20) patients before death, but only two patients (10, 2/20) were diagnosed by imaging studies before death. The rate of Goldman category major 1 was 13.2% (10/76) in 1999, 7.3% (4/55) in 2009 and 6.9% (8/116) in 2019. Although the rate of Goldman category major 1 appeared decreasing, the difference was not statistically significant. The autopsy rate was significantly higher in 2019 (8.4%, 116/1386) than in 2009 (4.4%, 55/1240). CONCLUSIONS: The incidence of PTE is uncommon. Despite the advances of the radiological techniques, radiological imaging studies did not detect the majority of PTEs. The discrepancy between the antemortem clinical and the postmortem diagnosis has not improved significantly over the past 30 years, emphasizing the value of autopsy.

FOXO3 regulates a common genomic program in aging and glioblastoma stem cells.

Background: Glioblastoma (GBM) is an aggressive, age-associated malignant glioma that contains populations of cancer stem cells. These glioma stem cells (GSCs) evade therapeutic interventions and repopulate tumors due to their existence in a slowly cycling quiescent state. Although aging is well known to increase cancer initiation, the extent to which the mechanisms supporting GSC tumorigenicity are related to physiological aging remains unknown. Aims: Here, we investigate the transcriptional mechanisms by which Forkhead Box O3 (FOXO3), a transcriptional regulator that promotes healthy aging, affects GSC function and the extent to which FOXO3 transcriptional networks are dysregulated in aging and GBM. Methods and results: We performed transcriptome analysis of clinical GBM tumors and observed that high FOXO3 activity is associated with gene expression signatures of stem cell quiescence, reduced oxidative metabolism, and improved patient outcomes. Consistent with these findings, we show that elevated FOXO3 activity significantly reduces the proliferation of GBM-derived GSCs. Using RNA-seq, we find that functional ablation of FOXO3 in GSCs rewires the transcriptional circuitry associated with metabolism, epigenetic stability, quiescence, and differentiation. Since FOXO3 has been implicated in healthy aging, we then investigated the extent to which it regulates common transcriptional programs in aging neural stem cells (NSCs) and GSCs. We uncover a shared transcriptional program and, most strikingly, find that FOXO3-regulated pathways are associated with altered mitochondrial functions in both aging and GBM. Conclusions: This work identifies a FOXO-associated transcriptional program that correlates between GSCs and aging NSCs and is enriched for metabolic and stemness pathways connected with GBM and aging.

Hypertrophy of the Anterior External Arcuate Fasciculus: A Rare Variant With Implications for the Development of the Arcuate Nucleus.

A rare anatomic variant of a markedly enlarged anterior external arcuate fasciculus (AEAF) on the ventral medullary surface is reported and compared to two controls. The hypertrophic AEAF was nine times larger in diameter than normal, whereas the arcuate nucleus (AN) and inferior olivary nucleus (ION) appeared histologically normal in size and neuronal distribution, and morphometric analysis of the AN confirmed that it was within the normal range. Calbindin-2 (calretinin, CALB2) expression was identified in the AN and in the fibers of the normal AEAF. The hypertrophic AEAF did not contain calbindin-2-expressing fibers. CALB2 expression was also present in the ventrolateral portion of the ION, both in the index case and in one of the control cases. The origin of the additional fibers was not identified; however, the potential origin of these fibers and its implications for the development of the AEAF are discussed.

Neuropathology Education Using Social Media.

Social media use continues to grow among pathologists. Discussions of current topics, posts of educational information, and images of pathological entities are commonly found and distributed on popular sites such as Facebook and Twitter. However, little is known about the presence of neuropathology content in social media and the audience for such content. We designed and distributed a survey to assess the demographics of users viewing neuropathology content and their opinions about neuropathology in social media. User posts on the Facebook group, Surgical Neuropathology, were also analyzed. The results show that there is a demand for neuropathology content of high quality, curated by experts, and that this demand is present among both specialists and nonspecialists. These findings suggest that social media may be useful for rapid dissemination of information in the field of neuropathology. This format also offers a unique opportunity to extend the reach of information to nonneuropathologists who may not receive neuropathology journals or have access to specialty-level neuropathology training, to build networks between professionals, and potentially to influence public opinion of neuropathology on an international scale.

Predicting survival of children with CNS tumors using proton magnetic resonance spectroscopic imaging biomarkers.

Using brain proton magnetic resonance spectroscopic imaging (MRSI) in children with central nervous system (CNS) tumors, we tested the hypothesis that combining information from biologically important metabolites, at diagnosis and prior to treatment, would improve prediction of survival. We evaluated brain proton MRSI exams in 76 children (median age at diagnosis: 74 months) with brain tumors. Important biomarkers, choline-containing compounds (Cho), N-acetylaspartate (NAA), total creatine (tCr), lipids and/or lactate (L), were measured at the "highest Cho region" and normalized to the tCr of surrounding healthy tissue. Neuropathological grading was performed using World Health Organization (WHO) criteria. Fifty-eight of 76 (76%) patients were alive at the end of the study period. The mean survival time for all subjects was 52 months. Univariate analysis demonstrated that Cho, L, Cho/NAA and tumor grade differed significantly between survivors and non-survivors (P< or =0.05). Multiple logistic regression and stepwise multivariate Cox regression indicated that Cho + 0.1L was the only independent predictor of survival (likelihood ratio test = 10.27, P<0.001; Cox regression, P=0.004). The combined index Cho + 0.1L was more accurate and more specific predictor than Cho or Cho/NAA. Accuracy and specificity for Cho + 0.1L were 80% and 86%, respectively. We conclude that brain proton MRSI biomarkers predict survival of children with CNS tumors better than does standard histopathology. More accurate prediction using this non-invasive technique represents an important advance and may suggest more appropriate therapy, especially when diagnostic biopsy is not feasible.

Atrophy of the tongue following complete versus partial hypoglossal nerve transection in a canine model.

OBJECTIVES/HYPOTHESIS: The hypoglossal nerve (XII) has been used as a donor nerve in facial and laryngeal reinnervation. The purpose of this study was to investigate the neuromuscular changes that occur within the tongue following partial or complete transection of XII using a canine model. STUDY DESIGN: Histopathological comparison of tongue denervation following two types of XII resection in a canine model. METHODS: Ten adult canines underwent complete unilateral resection of XII or resection of only the medial terminal branch of the hypoglossal nerve (mXII). After 6 months of recovery, tongue specimens were analyzed histopathologically using whole cross-sections. Routine histologic sections were assessed by two neuropathologists blinded to the type of denervation. The cross-sectional area was calculated of both sides of the tongue, and the amount of myosin was quantified morphometrically using immunohistochemistry for myosin (antimyosin heavy chain, fast isotype). Statistical comparison between partial and complete denervation was performed using the Student t test. RESULTS: Six months following XII transection, quantitative measures of the cross-sectional area of the tongue and content of myosin demonstrated severe muscle atrophy on the operated side of the tongue for both groups, compared to the nonoperated side. For partial transection involving only mXII, the degree of atrophy was less severe (P < .05). CONCLUSIONS: This study provides new histological information demonstrating that partial resection of the hypoglossal nerve, sacrificing only the proximal medial branch of the hypoglossal nerve (mXII), results in less severe atrophy of the tongue than complete transection of the entire hypoglossal nerve. LEVEL OF EVIDENCE: NA Laryngoscope, 126:2689-2693, 2016.

Proliferative potential of human astrocytes.

Although a number of studies have demonstrated proliferation of nonneoplastic astrocytes in experimental animal models, the proliferative potential of human astrocytes has not been well defined. Using double-label immunohistochemistry, we identified proliferating cells with the proliferation marker MIB-1 and astrocytes with glial fibrillary acidic protein staining in human biopsy and autopsy tissue. MIB-1 labeling of astrocytes was monitored in a variety of conditions containing significant numbers of reactive astrocytes, including infections, arteriovenous malformations, demyelinating lesions, metastatic tumors, and long-standing gliosis. Twenty-nine of a total of 54 cases showed no evidence of astrocyte-specific MIB-1 labeling despite prominent reactive changes. An average proliferation rate of 0.9% was present in the remaining 25 cases. Labeling indices were highest in infectious conditions and acute demyelinating lesions. We also examined astrocyte proliferation in 5 cases of progressive multifocal leukoencephalopathy. Astrocytic labeling indices were notably elevated in these cases, with an average labeling index of 5.8%. We conclude that low, but appreciable, astrocytic proliferation may occur in nonneoplastic human astrocytes. These findings have implications for astrocyte function in the normal and disease states and for the diagnostic distinction between reactive lesions and low-grade astrocytic neoplasms.

Noninvasive magnetic resonance spectroscopic imaging biomarkers to predict the clinical grade of pediatric brain tumors.

The diagnosis and therapy of childhood brain tumors, most of which are low grade, can be complicated because of their frequent adjacent location to crucial structures, which limits diagnostic biopsy. Also, although new prognostic biomarkers identified by molecular analysis or DNA microarray gene profiling are promising, they too depend on invasive biopsy. Here, we test the hypothesis that combining information from biologically important intracellular molecules (biomarkers), noninvasively obtained by proton magnetic resonance spectroscopic imaging, will increase the diagnostic accuracy in determining the clinical grade of pediatric brain tumors. We evaluate the proton magnetic resonance spectroscopic imaging exams for 66 children with brain tumors. The intracellular biomarkers for choline-containing compounds (Cho), N-acetylaspartate, total creatine, and lipids and/or lactate were measured at the highest Cho region and normalized to the surrounding healthy tissue total creatine. Neuropathological grading was done with WHO criteria. Normalized Cho and lipids and/or lactate were elevated in high-grade (n = 23) versus low-grade (n = 43) tumors, which multiple logistic regression confirmed are independent predictors of tumor grade (for Cho, odds ratio 24.8, P < 0.001; and for lipids and/or lactate, odds ratio 4.4, P < 0.001). A linear combination of normalized Cho and lipids and/or lactate that maximizes diagnostic accuracy was calculated by maximizing the area under the receiver operating characteristic curve. Proton magnetic resonance spectroscopic imaging, although not a proxy for histology, provides noninvasive, in vivo biomarkers for predicting clinical grades of pediatric brain tumors.

Identification of Global DNA Methylation Signatures in Glioblastoma-Derived Cancer Stem Cells.

Glioblastoma (GBM) is the most common and most aggressive primary brain tumor in adults. The existence of a small population of stem-like tumor cells that efficiently propagate tumors and resist cytotoxic therapy is one proposed mechanism leading to the resilient behavior of tumor cells and poor prognosis. In this study, we performed an in-depth analysis of the DNA methylation landscape in GBM-derived cancer stem cells (GSCs). Parallel comparisons of primary tumors and GSC lines derived from these tumors with normal controls (a neural stem cell (NSC) line and normal brain tissue) identified groups of hyper- and hypomethylated genes that display a trend of either increasing or decreasing methylation levels in the order of controls, primary GBMs, and their counterpart GSC lines, respectively. Interestingly, concurrent promoter hypermethylation and gene body hypomethylation were observed in a subset of genes including MGMT, AJAP1 and PTPRN2. These unique DNA methylation signatures were also found in primary GBM-derived xenograft tumors indicating that they are not tissue culture-related epigenetic changes. Integration of GSC-specific epigenetic signatures with gene expression analysis further identified candidate tumor suppressor genes that are frequently down-regulated in GBMs such as SPINT2, NEFM and PENK. Forced re-expression of SPINT2 reduced glioma cell proliferative capacity, anchorage independent growth, cell motility, and tumor sphere formation in vitro. The results from this study demonstrate that GSCs possess unique epigenetic signatures that may play important roles in the pathogenesis of GBM.

Neuroimaging in pediatric brain tumors: Gd-DTPA-enhanced, hemodynamic, and diffusion MR imaging compared with MR spectroscopic imaging.

BACKGROUND AND PURPOSE: Gadolinium-enhanced MR images assist in defining tumor borders; however, the relation between tumor cell extent and contrast-enhanced regions is unclear. Our aim was to improve conventional neuroimaging of pediatric brain tumors with hemodynamic, diffusion, and spectroscopic MR imaging. METHODS: We performed conventional MR and MR spectroscopic imaging in 31 children with neuroglial brain tumors. Hemodynamic MR imaging was performed in 16 patients with a first-pass intravenous bolus of gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA); apparent diffusion coefficients (ADCs) were measured in 12 patients. To account for multiple measurements in a patient, we used a nested analysis of variance. RESULTS: At MR spectroscopy, choline (Cho)-containing compounds (indicating tumor) and lipid levels (indicating necrosis) did not correlate with percent Gd-DTPA enhancement on MR images. Percent enhancement was positively correlated with relative cerebral blood volumes (rCBVs) (P =.05) and negatively correlated with ADCs (P <.001). Stepwise multiple linear regression revealed that rCBV (P =.008), ADC (P =.022), and lipid (P <.001) levels were significant independent predictors of percent enhancement. Tumor spectral patterns were detected in tumor regions and outside enhancing tumor beds in patients with clinical progression; these were confirmed at neuropathologic analysis. CONCLUSION: MR spectroscopic imaging improves the assessment of pediatric brain tumors by adding biochemical information regarding tumor involvement and by depicting residual or recurrent tumor outside the Gd-DTPA-enhanced tumor bed. rCBV and ADC mapping complemented MR spectroscopic imaging. We recommend the use of MR spectroscopic imaging in addition to conventional MR imaging in assessing pediatric brain tumors.

Congenital cystic eye: recurrence after initial surgical removal.

Congenital cystic eye arises from failure of invagination of the primary optic vesicle. The cyst lacks the usual intraocular structures and contains only abortive derivatives of the anterior and posterior portions of the primary optic vesicle. Because the cystic eye usually occupies a large portion of the orbit and distends the upper eyelid, surgical excision is often carried out. We report a case of congenital cystic eye in which an initial attempt at excision was followed in three months by recurrence of the cyst in the orbit. A second excision appears to have eliminated the cyst since no recurrence has become evident in the ensuing two years. The pathology of the cyst from both excision specimens suggests that the pigmented cells from the posterior portion of the cyst were not included in the first resection. Based on this experience, we conclude that every effort should be made to excise all of the congenital cystic eye when surgical removal is undertaken.

Biochemical characterization of pediatric brain tumors by using in vivo and ex vivo magnetic resonance spectroscopy.

OBJECT: Magnetic resonance (MR) spectroscopy provides biochemical information about tumors. The authors sought to determine the relationship between in vivo and ex vivo biochemical characterization of pediatric brain tumors by using MR spectroscopy. Their hypothesis was that ex vivo MR spectroscopy provides a link between in vivo MR spectroscopy and neuropathological analysis. METHODS: In vivo proton MR spectroscopy was performed before surgery in 11 patients with neuroepithelial tumors. During resection, a total of 40 tumor biopsy samples were obtained from within the volume of interest identified on in vivo MR spectroscopy and were frozen immediately in liquid nitrogen. High-Resolution Magic Angle Spinning (HRMAS) was used to perform ex vivo MR spectroscopy in these 40 tumor biopsy samples. Neuropathological analysis was performed using the same biopsy samples, and the tumors were classified as ependymoma, choroid plexus carcinoma, pineoblastoma (one each), and pilocytic astrocytoma, medullobastoma, low-grade glioma, and glioblastoma multiforme (two each). Ex vivo HRMAS MR spectroscopy improved line widths and line shapes in the spectra, compared with in vivo MR spectroscopy. Choline (Cho) detected in vivo corresponded to three different peaks ex vivo (glycerophosphocholine, phosphocholine [PCho], and Cho). Metabolite ratios from in vivo spectra correlated with ratios from ex vivo spectra (Pearson correlation coefficient range r = 0.72-0.91; p < or = 0.01). Metabolite ratios from ex vivo spectra, such as PCho/ total creatine (tCr) and lipid/tCr, correlated with the percentage of cancerous tissue and percentage of tumor necrosis, respectively (r = 0.84; p < 0.001). CONCLUSIONS: Agreement between in vivo and ex vivo MR spectroscopy indicates that ex vivo HRMAS MR spectroscopy can improve resolution of this modality and provide a link between in vivo MR spectroscopy and neuropathological analysis.

Neurologic and histopathologic evaluation after high-volume intrathecal amitriptyline.

BACKGROUND AND OBJECTIVES: Accumulating evidence indicates that amitriptyline decreases pain sensation when administered orally, intraperitoneally, or for sciatic nerve block. Previous reports of intrathecal administration of amitriptyline have yielded inconsistent results. The failure of amitriptyline to provide antinociception may partly be related to its high logP (octanol-water partition coefficient) and consequent poor spread within the cerebrospinal fluid. We evaluated spinal block after various concentrations of amitriptyline administered intrathecally in a fixed high volume. METHODS: We administered 100 microL of 5, 10, 15.9 (0.5%), 25, 50, or 100 mmol/L amitriptyline hydrochloride solution or 100 microL of 15.4 mmol/L (0.5%) bupivacaine hydrochloride solution intrathecally to rats. The neurologic deficit was evaluated by antinociceptive, motor, and proprioceptive responses, and the spinal cord was examined for histopathologic changes. RESULTS: Doses of 100 microL amitriptyline at 15.9 mmol/L (0.5%) and 25 mmol/L produced longer complete nerve block than did bupivacaine at 15.4 mmol/L (0.5%); 5 and 10 mmol/L amitriptyline produced only partial nerve block. However, with 100 microL intrathecal amitriptyline at 50 and 100 mmol/L, many rats did not fully recover from spinal block. Severe axonal degeneration, myelin breakdown, and replacement of neuronal structures by vacuoles were seen in the spinal root section of animals injected with concentrations higher than 25 mmol/L amitriptyline. CONCLUSIONS: At lower doses, intrathecal injection of high volumes of amitriptyline results in long-acting spinal block. At higher doses, intrathecal amitriptyline results in irreversible neurologic deficit. Therefore, we do not recommend the use of intrathecal amitriptyline because of a very low therapeutic index.

Histological quantitation of brain injury using whole slide imaging: a pilot validation study in mice.

Quantitative assessment of serial brain sections provides an objective measure of neurological events at cellular and molecular levels but is difficult to implement in experimental neuroscience laboratories because of variation from person-to-person and the time required for analysis. Whole slide imaging (WSI) technology, recently introduced for pathological diagnoses, offers an electronic environment and a variety of computational tools for performing high-throughput histological analysis and managing the associated information. In our study, we applied various algorithms to quantify histologic changes associated with brain injury and compared the results to manual assessment. WSI showed a high degree of concordance with manual quantitation by Pearson correlation and strong agreement using Bland-Altman plots in: (i) cortical necrosis in cresyl-violet-stained brain sections of mice after focal cerebral ischemia; (ii) intracerebral hemorrhage in ischemic mouse brains for automated annotation of the small regions, rather than whole hemisphere of the tissue sections; (iii) Iba1-immunoreactive cell density in the adjacent and remote brain regions of mice subject to controlled cortical impact (CCI); and (iv) neuronal degeneration by silver staining after CCI. These results show that WSI, when appropriately applied and carefully validated, is a highly efficient and unbiased tool to locate and identify neuropathological features, delineate affected regions and histologically quantify these events.

Inhibition of prostate cancer bone metastasis by synthetic TF antigen mimic/galectin-3 inhibitor lactulose-L-leucine.

Currently incurable, prostate cancer metastasis has a remarkable ability to spread to the skeleton. Previous studies demonstrated that interactions mediated by the cancer-associated Thomsen-Friedenreich glycoantigen (TF-Ag) and the carbohydrate-binding protein galectin-3 play an important role in several rate-limiting steps of cancer metastasis such as metastatic cell adhesion to bone marrow endothelium, homotypic tumor cell aggregation, and clonogenic survival and growth. This study investigated the ability of a synthetic small-molecular-weight nontoxic carbohydrate-based TF-Ag mimic lactulose-L-leucine (Lac-L-Leu) to inhibit these processes in vitro and, ultimately, prostate cancer bone metastasis in vivo. Using an in vivo mouse model, based on intracardiac injection of human PC-3 prostate carcinoma cells stably expressing luciferase, we investigated the ability of Lac-L-Leu to impede the establishment and growth of bone metastasis. Parallel-flow chamber assay, homotypic aggregation assay, modified Boyden chamber assay, and clonogenic growth assay were used to assess the effects of Lac-L-Leu on tumor cell adhesion to the endothelium, homotypic tumor cell aggregation, transendothelial migration, and clonogenic survival and growth, respectively. We report that daily intraperitoneal administration of Lac-L-Leu resulted in a three-fold (P < .05) decrease in metastatic tumor burden compared with the untreated control. Mechanistically, the effect of Lac-L-Leu, which binds and inhibits galectins by mimicking essential structural features of the TF-Ag, was associated with a dose-dependent inhibition of prostate cancer cell adhesion to bone marrow endothelium, homotypic aggregation, transendothelial migration, and clonogenic growth. We conclude that small-molecular-weight carbohydrate-based compounds targeting ß-galactoside-mediated interactions could provide valuable means for controlling and preventing metastatic prostate cancer spread to the skeleton.