Jason K. Wasserman

Minocycline protects the blood-brain barrier and reduces edema following intracerebral hemorrhage in the rat

Intracerebral hemorrhage (ICH) results from rupture of a blood vessel in the brain. After ICH, the blood-brain barrier (BBB) surrounding the hematoma is disrupted, leading to cerebral edema. In both animals and humans, edema coincides with inflammation, which is characterized by production of pro-inflammatory cytokines, activation of resident brain microglia and migration of peripheral immune cells into the brain. Accordingly, inflammation is an attractive target for reducing edema following ICH. In the present study, BBB damage was assessed by quantifying intact microvessels surrounding the hematoma, monitoring extravasation of IgG and measuring brain water content 3 days after ICH induced by collagenase injection into the rat striatum. In the injured brain, the water content increased in both ipsilateral and contralateral hemispheres compared with the normal brain. Quantitative real-time RT-PCR revealed an up-regulation of inflammatory genes associated with BBB damage; IL1beta, TNFalpha and most notably, MMP-12. Immunostaining showed MMP-12 in damaged microvessels and their subsequent loss from tissue surrounding the hematoma. MMP-12 was also observed for the first time in neurons. Dual-antibody labeling demonstrated that neutrophils were the predominant source of TNFalpha protein. Intraperitoneal injection of the tetracycline derivative, minocycline, beginning 6 h after ICH ameliorated the damage by reducing microvessel loss, extravasation of plasma proteins and edema; decreasing TNFalpha and MMP-12 expression; and reducing the numbers of TNFalpha-positive cells and neutrophils in the brain. Thus, minocycline, administered at a clinically relevant time, appears to target the inflammatory processes involved in edema development after ICH.

Evolution of the inflammatory response in the brain following intracerebral hemorrhage and effects of delayed minocycline treatment

There are no effective treatments for intracerebral hemorrhage (ICH). Although inflammation is a potential therapeutic target, there is a dearth of information about time-dependent and cell-specific changes in the expression of inflammation-related genes. Using the collagenase-induced ICH model in rats and real-time quantitative RT-PCR we monitored mRNA levels of markers of glial activation, pro- and anti-inflammatory cytokines, enzymes responsible for cytokine activation and several matrix metalloproteases at 6 h and 1, 3 and 7 days after ICH onset. For the most highly up-regulated genes, immunohistochemistry was then used to identify cell-specific protein expression. Finally, minocycline, a drug widely reported to reduce damage in several models of brain injury, was used to test the hypothesis that it can reduce up-regulation of inflammation-related genes when administered using a clinically relevant dosing regime: intraperitoneal injection beginning 6 h after ICH. Our results show a complex inflammatory response, with different brain cell types producing several pro- and anti-inflammatory molecules for at least 7 days after ICH onset. Included is the first demonstration that astrocytes are an important source of interleukin-1beta (IL-1beta), interleukin-1 receptor antagonist (IL-1ra), interleukin-6 (IL-6) and MMP-12. Importantly, our results demonstrate that while delayed minocycline treatment effectively reduces early up-regulation of TNFalpha and MMP-12, its efficacy is lost when treatment is extended for up to a week, and it does not reduce several other genes associated with microglia activation. These results suggest caution in extrapolating to ICH the promising results of minocycline treatment in other models of brain injury.

Neuron death and inflammation in a rat model of intracerebral hemorrhage: Effects of delayed minocycline treatment

After intracerebral hemorrhage (ICH), blood entry is followed by neuron death and an inflammatory response, but development of pharmacological therapies has been hampered by an inadequate understanding of the spatial and temporal relationship between neuron death and inflammation. Using a rat model of ICH, we first investigated these relationships at 6 h, and 1, 3 and 7 days. At the edge of the hematoma, no degenerating neurons were observed at 6 h; however, dying neurons were present between 1 and 3 days, with peak neuron death occurring at 1 day. This is apparently the first report of ongoing neuron death at the edge of the hematoma during a time window that is appropriate for human therapy. Neuron death was limited to the edge of the hematoma, with no degenerating neurons in the striatum surrounding the hematoma, despite robust and prolonged microglia activation. Importantly, neuron loss at the edge of the hematoma was spatially and temporally associated with accumulation and activation of microglia/macrophages. We then tested the hypothesis that treatment with the tetracycline derivative, minocycline, after the hematoma had reached a maximal size, will reduce inflammation and neuron damage. Minocycline injection (45 mg/kg i.v. at 6 h, and i.p. at 24, 48 and 72 h) failed to reduce neuron loss outside the hematoma or striatal tissue loss (assessed at 7 days), despite reducing the number of neutrophils and activated microglia/macrophages. Thus, minocycline does not appear to target the mechanisms responsible for cell death in this model of ICH.

Glial responses, neuron death and lesion resolution after intracerebral hemorrhage in young vs. aged rats

Intracerebral hemorrhage (ICH) usually affects older humans but almost no experimental studies have assessed aged animals. We address how aging alters inflammation, neuron death and lesion resolution after a hemorrhage in the rat striatum. In the normal aged brain, microglia displayed a ‘dystrophic’ phenotype, with shorter cellular processes and large gaps between adjacent cells, and there was more astrocyte reactivity. The ICH injury was monitored as hematoma volume and number of dying neurons at 1 and 3 days, and the volume of the residual lesion, ventricles and lost tissue at 28 days. Inflammation at 1 and 3 days was assessed from densities of microglia with resting vs. activated morphologies, or expressing the lysosomal marker ED1. Despite an initial delay in neuron death in aged animals, by 28 days, there was no difference in neuron density or volume of tissue lost. However, lesion resolution was impaired in aged animals and there was less compensatory ventricular expansion. At 1 day after ICH, there were fewer activated microglia/macrophages in the aged brain, but by 3 days there were more of these cells at the edge of the hematoma and in the surrounding parenchyma. In both age groups a glial limitans had developed by 3 days, but astrocyte reactivity and the spread of activated microglia/macrophages into the surrounding parenchyma was greater in the aged. These findings have important implications for efforts to reduce secondary injury after ICH and to develop anti‐inflammatory therapies to treat ICH in aged humans.

Stratified, Urgent Care for Transient Ischemic Attack Results in Low Stroke Rates

Background and Purpose— Transient ischemic attack (TIA) is a marker for early risk of stroke. No previous studies have assessed the use of urgent stroke prevention clinics for emergency department (ED) patients with TIA. We hypothesized that an ABCD2-based ED triaging tool for TIA with outpatient management would be associated with lower 90-day stroke rate than that predicted by ABCD2. Methods— A cohort of prospectively identified patients presenting with symptoms suggestive of TIA seen in 2 tertiary-care EDs. These patients were divided into 3 strata based on their ACBD2 score, and triage targets were set for each stratum. All patients received the same standard of care in the Stroke Clinic regardless of their risk score. Primary outcome was stroke by 90 days of index TIA. Secondary outcomes were subsequent TIA, myocardial infarction, or death. Results— One-thousand ninety-three patients met the inclusion criteria; 982 patients completed 90-day follow-up and comprised the final cohort. After stratification, 32%, 49%, and 19% of patients were categorized as low-, moderate-, or high-risk, respectively. The overall 90-day risk of stroke in all patients was 3.2%, compared with the ABCD2-predicted risk of 9.2%. Only 1.6% of patients with TIA/minor stroke were admitted from the ED. The risk of subsequent TIA, myocardial infarction, or death by 90 days was 5.5%, 0.1%, and 1.7%, respectively. Conclusion— Outpatient care in a rapid-access stroke prevention clinic using the ABCD2 score for triage resulted in a low 90-day stroke rate for patients in the ED with TIA. Benefit occurred without requiring admission for most patients.

White matter injury in young and aged rats after intracerebral hemorrhage

Experimental studies of intracerebral hemorrhage (ICH) have focused on neuron death, with little or no information on axonal and myelin damage outside the hematoma. Because development of effective therapies will require an understanding of white matter injury, we examined white matter injury and its spatial and temporal relationship with microglial/macrophage activation in a collagenase model of rat striatal ICH. The hematoma and parenchyma surrounding the hematoma were assessed in young and aged animals at 6 h, 1, 3 and 28 days after ICH onset. Demyelination occurred inside and at the edge of the hematoma; regions where we have shown substantial neuron death. In contrast, there was axonal damage without demyelination at the edge of the hematoma, and by 3 days this damage had spread to the surrounding parenchyma, a region where we have shown there is no neuron death. Because the axonal damage preceded infiltration of activated microglia into the white matter tracts (seen at 3 days), our results support the hypothesis that these cells respond to, rather than perpetrate the damage. Importantly, axonal damage was worse in aged animals, which provides a plausible explanation for the poorer functional recovery of older animals after ICH, despite a similar loss of grey matter. Our findings support strategies that target white matter injury to reduce neurological impairment after ICH.

Evolution of Computed Tomography Angiography Spot Sign Is Consistent With a Site of Active Hemorrhage in Acute Intracerebral Hemorrhage

Background and Purpose— CT angiography spot sign predicts hematoma expansion in patients with acute intracerebral hemorrhage (ICH). The spot sign may represent a site of active extravasation, a locus of arrested hemorrhage forming fibrin globes, or represent associated epiphenomena such as hypertensive microaneurysms. We sought to describe the evolution of spot signs over 60 seconds in acute ICH using dynamic CT angiography and determine whether they grow and diffuse into the hematoma as would be expected with active extravasation. Methods— We prospectively identified consecutive patients presenting with spontaneous ICH <6 hours from symptom onset that completed dynamic CT angiography imaging over a 60-second acquisition protocol. We determined spot positivity, quantified spot volumes, and then used repeated-measures ANOVA to assess changes in spot volume over time. Results— We collected data on 35 patients; 13 of 35 (37%) patients were spot-positive. Spot-positive patients had larger median ICH volume compared with spot-negative patients (median 10.7 versus 49.2 mL; P=0.007). Maximal spot sign volumes ranged from 0.02 to 2.8 mL (median 0.17 mL). Spot sign volumes increased significantly with time (P<0.001) and seemed to disperse into the hematoma in all cases. Three of 13 (23%) spot-positive patients presented with 2 distinct spot signs, but the remaining patients either had only 1 spot sign or different contiguous components of an irregularly shaped spot sign. Conclusions— In this dynamic CT angiography study of ICH, spot signs evolve consistent with sites of active extravasation.

Immunohistochemistry in the Diagnosis of Mucinous Neoplasms Involving the Ovary: The Added Value of SATB2 and Biomarker Discovery Through Protein Expression Database Mining.

Immunohistochemistry is frequently used to identify ovarian mucinous neoplasms as primary or metastatic; however, there is significant overlap in expression patterns. We compared traditional markers (CK7, CK20, CDX2, PAX8, estrogen receptor, &bgr;-catenin, MUC1, MUC2, and MUC5AC) to 2 novel proteins identified through mining of the Human Protein Atlas expression database: SATB2 and POF1B. The study cohort included 49 primary gastrointestinal (GI) mucinous adenocarcinomas (19 colorectal, 15 gastric, 15 pancreatobiliary), 60 primary ovarian mucinous neoplasms (19 cystadenomas, 21 borderline tumors, 20 adenocarcinomas), and 19 metastatic carcinomas to the ovary (14 lower and 5 upper GI primaries). Immunohistochemistry was performed on tissue microarrays, scored and interpreted as negative (absent or focal/weak) or positive. Metastatic tumors were frequently unilateral (42.8% of tumors from lower and 40% of tumors from upper tract) and ≥10 cm (85.7% of tumors from lower and 80% of tumors from upper tract). CK7 was positive in 88.5% upper GI and 88.3% primary ovarian compared with 24.3% lower GI neoplasms. CK20 and CDX2 were positive in 84.8% and 100% of lower GI tumors, respectively; however, expression was also common in upper GI (CK20 42.8%, CDX2 50%) and primary ovarian neoplasms (CK20 65.7%, CDX2 38.3%). Conversely, SATB2 was more specific for lower GI origin, being positive in 78.8% lower GI but only 11.5% upper GI and 1.7% primary ovarian neoplasms. PAX8 expression was common in primary ovarian neoplasms (75% of all neoplasms, 65% of carcinomas); only 1 (1.5%) GI tumor was positive. MUC2 and &bgr;-catenin were frequently positive in lower GI tumors (96.9% and 51.5%, respectively). Estrogen receptor expression was only seen in primary ovarian neoplasms (13.3%). Nuclear premature ovarian failure 1B (POF1B) expression was seen in malignant tumors regardless of their origin. A panel including CK7, SATB2, and PAX8 separated primary from secondary GI neoplasms with up to 77.1% sensitivity and 99% specificity, outperforming tumor laterality and size. Second-line markers such as CDX2, MUC2, estrogen receptor, MUC1, and &bgr;-catenin increased the sensitivity of immunohistochemistry in excluding lower GI origin. Biomarker search using proteomic databases has a value in diagnostic pathology, as shown with SATB2; however, as seen with POF1B, expression profiles in these databases are not always reproduced in larger cohorts.

Clinical and pathological features of intraductal papillary neoplasm of the biliary tract and gallbladder

BACKGROUND Intraductal papillary neoplasms of the biliary tract (IPNB) and intracholecystic papillary neoplasms (ICPN) are rare tumours characterized by intraluminal papillary growth that can be associated with invasive carcinoma. Their natural history remains poorly understood. This study examines clinicopathological features and outcomes. METHODS Patients who underwent surgery for IPNB/ICPN (2008-2014) were identified. Descriptive statistics and survival data were generated. RESULTS Of 23 patients with IPNB/ICPN, 10 were male, and the mean age was 68 years. The most common presentations were abdominal pain (n = 10) and jaundice (n = 9). Tumour locations were: intrahepatic (n = 5), hilar (n = 3), the extrahepatic bile duct (n = 8) and the gallbladder (n = 7). Invasive cancer was found in 20/23 patients. Epithelial subtypes included pancreatobiliary (n = 15), intestinal (n = 7) and gastric (n = 1). The median follow-up was 30 months. The 5-year overall (OS) and disease-free survivals (DFS) were 51% and 57%, respectively. Decreased OS (P = 0.09) and DFS (P = 0.05) were seen in patients with tumours expressing MUC1 on immunohistochemistry (IHC). CONCLUSION IPNB/ICPN are rare precursor lesions that can affect the entire biliary epithelium. At pathology, the majority of patients have invasive carcinoma, thus warranting a radical resection. Patients with tumours expressing MUC1 appear to have worse OS and DFSs.

Differentiated Squamous Intraepithelial Neoplasia Associated with Squamous Cell Carcinoma of The Anal Canal

Differentiated squamous intraepithelial neoplasia (DSIN) has been described in several sites, including the upper aerodigestive tract and vulva, so this study investigated whether it also occurred in the anal canal.