Photoacoustic Imaging for Assessing Tissue Oxygenation Changes in Rat Hepatic Fibrosis.

Chronic liver inflammation progressively evokes fibrosis and cirrhosis resulting in compromised liver function, and often leading to cancer. Early diagnosis and staging of fibrosis is crucial because the five-year survival rate of early-stage liver cancer is high. This study investigates the progression of hepatic fibrosis induced in rats following ingestion of diethylnitrosamine (DEN). Changes in oxygen saturation and hemoglobin concentration resulting from chronic inflammation were assayed longitudinally during DEN ingestion by photoacoustic imaging (PAI). Accompanying liver tissue changes were monitored simultaneously by B-mode sonographic imaging. Oxygen saturation and hemoglobin levels in the liver increased over 5 weeks and peaked at 10 weeks before decreasing at 13 weeks of DEN ingestion. The oxygenation changes were accompanied by an increase in hepatic echogenicity and coarseness in the ultrasound image. Histology at 13 weeks confirmed the development of severe fibrosis and cirrhosis. The observed increase in PA signal representing enhanced blood oxygenation is likely an inflammatory physiological response to the dietary DEN insult that increases blood flow by the development of neovasculature to supply oxygen to a fibrotic liver during the progression of hepatic fibrosis. Assessment of oxygenation by PAI may play an important role in the future assessment of hepatic fibrosis.

Dose-dependent effects of ultrasound therapy on hepatocellular carcinoma.

Non-invasive ischemic cancer therapy requires reduced blood flow whereas drug delivery and radiation therapy require increased tumor perfusion for a better response. In this study we investigate the hypothesis that different dose models of antivascular ultrasound therapy (AVUS) can have opposite effects on hepatocellular carcinoma (HCC) tumor blood flow. HCC was induced in 22 Wistar rats by ingestion of diethylnitrosamine (DEN) for 12 weeks. Rats received AVUS treatment at low and high doses. Low dose group received 1 watt/cm2 ultrasound for 1 min with 0.2 mL microbubbles injected IV. High dose group received 2 watts/cm2 for 2 min with 0.7 mL microbubbles IV. A sham group did not receive any treatment. Tumor perfusion was measured before and after AVUS with contrast-enhanced ultrasound. Quantitative perfusion measures: perfusion index (PI) and peak enhancement (PE) were obtained from each AVUS dose. After high-dose AVUS, PE and PI decreased by an average of 58.1 ± 4.9% and 49.1 ± 6.5 % respectively. Conversely, following low dose AVUS, PE and PI increased from baseline by an average of 47.8 ± 4.5% % and 20.3 ± 2.4 %, respectively. The high-dose AVUS therapy decreased tumoral perfusion, an effect that could be used for noninvasive ischemic therapy. Conversely, low-dose therapy increased tumor perfusion, which may improve drug delivery or radiation therapy. These opposite therapy effects can support multiple roles for AVUS in cancer therapy by tunable modulation of blood flow in tumors.

Microbubble-enhanced ultrasound for the antivascular treatment and monitoring of hepatocellular carcinoma.

Background and Objective: Hepatocellular carcinoma (HCC) is the most common primary liver malignancy, and its current management relies heavily on locoregional therapy for curative therapy, bridge to transplant, and palliative therapy. Locoregional therapies include ablation and hepatic artery therapies such as embolization and radioembolization. In this study we evaluate the feasibility of using novel antivascular ultrasound (AVUS) as a noninvasive locoregional therapy to reduce perfusion in HCC lesions in a rat model and, monitor the effect with contrast-enhanced ultrasound imaging. Methods: HCC was induced in 36 Wistar rats by the ingestion of 0.01% diethylnitrosamine (DEN) for 12 weeks. Two therapy regimens of AVUS were evaluated. A primary regimen (n = 19) utilized 2-W/cm2, 3-MHz ultrasound (US) for 6 minutes insonation with 0.7 ml of microbubbles administered as an intravenous bolus. An alternate dose at half the primary intensity, sonication time, and contrast concentration was evaluated in 11 rats to assess the efficacy of a reduced dose. A control group (n = 6) received a sham therapy. Tumor perfusion was measured before and after AVUS with nonlinear contrast ultrasound (NLC) and power Doppler (PD). The quantitative perfusion measures included perfusion index (PI), peak enhancement (PE), time to peak (TTP), and perfusion area from NLC and PD scans. Total tumor area perfused during the scan was measured by a postprocessing algorithm called delta projection. Tumor histology was evaluated for signs of tissue injury and for vascular changes using CD31 immunohistochemistry. Results: DEN exposure induced autochthonous hepatocellular carcinoma lesions in all rats. Across all groups prior to therapy, there were no significant differences in the nonlinear contrast observations of peak enhancement and perfusion index. In the control group, there were no significant differences in any of the parameters after sham treatment. After the primary AVUS regimen, there were significant changes in all parameters (p ≤ 0.05) indicating substantial decreases in tumor perfusion. Peak enhancement in nonlinear contrast scans showed a 37.9% ± 10.1% decrease in tumor perfusion. Following reduced-dose AVUS, there were no significant changes in perfusion parameters, although there was a trend for the nonlinear contrast observations of peak enhancement and perfusion index to increase. Conclusion: This study translated low-intensity AVUS therapy into a realistic in vivo model of HCC and evaluated its effects on the tumor vasculature. The primary dose of AVUS tested resulted in significant vascular disruption and a corresponding reduction in tumor perfusion. A reduced dose of AVUS, on the other hand, was ineffective at disrupting perfusion but demonstrated the potential for enhancing tumor blood flow. Theranostic ultrasound, where acoustic energy and microbubbles are used to monitor the tumor neovasculature as well as disrupt the vasculature and treat lesions, could serve as a potent tool for delivering noninvasive, locoregional therapy for hepatocellular carcinoma.

B-mode ultrasound for the assessment of hepatic fibrosis: a quantitative multiparametric analysis for a radiomics approach.

Hepatic fibrosis and cirrhosis are a growing global health problem with increasing mortality rates. Early diagnosis and staging of hepatic fibrosis represent a major challenge. Currently liver biopsy is the gold standard for fibrosis assessment; however, biopsy requires an invasive procedure and is prone to sampling error and reader variability. In the current study we investigate using quantitative analysis of computer-extracted features of B-mode ultrasound as a non-invasive tool to characterize hepatic fibrosis. Twenty-two rats were administered diethylnitrosamine (DEN) orally for 12 weeks to induce hepatic fibrosis. Four control rats did not receive DEN. B-mode ultrasound scans sampling throughout the liver were acquired at baseline, 10, and 13 weeks. Computer extracted quantitative parameters representing brightness (echointensity, hepatorenal index) and variance (heterogeneity, anisotropy) of the liver were studied. DEN rats showed an increase in echointensity from 37.1 ± SD 7.8 to 53.5 ± 5.7 (10 w) to 57.5 ± 6.1 (13 w), while the control group remained unchanged at an average of 34.5 ± 4.5. The three other features studied increased similarly over time in the DEN group. Histologic analysis showed METAVIR fibrosis grades of F2-F4 in DEN rats and F0-F1 in controls. Increasing imaging parameters correlated with increasing METAVIR grades, and anisotropy showed the strongest correlation (ρ = 0.58). Sonographic parameters combined using multiparametric logistic regression were able to differentiate between clinically significant and insignificant fibrosis. Quantitative B-mode ultrasound imaging can be implemented in clinical settings as an accurate non-invasive tool for fibrosis assessment.

Hepatic Hemangiosarcoma in a Cynomolgus Macaque (Macaca fascicularis).

An experimentally naïve, 9-y-old, intact male cynomolgus macaque was reported for bleeding from an unidentified site. Sedated physical examination indicated mild gingival separation from the lingual aspect of the upper right canine tooth as the source of the hemorrhage. Physical exam revealed a firm mass adhered to the left zygomatic arch, 2 subcutaneous nodules on the chest, and a large mass in the cranial abdomen. Radiographs revealed a large soft-tissue mass in the cranial abdomen and multifocal nodules in the caudal lung fields. On ultrasonography, the liver was grossly enlarged and contained a cavi- tated mass. Hematology and serum chemistry results demonstrated severe regenerative anemia with normal clotting times and adequate platelet count. For humane reasons, euthanasia was elected. On gross examination, the liver was markedly enlarged by an expansile mass primarily affecting the median lobe, with multiple, smaller nodules throughout the remaining lobes. Multifocal round, firm nodules were observed on the surface of all lung lobes and throughout the omentum. Histologic examination of the hepatic, cutaneous, osseous, and pulmonary lesions demonstrated well-defined, endothelium-lined vascular channels arranged in cords with abundant hemorrhage; endothelial-cell immunomarkers confirmed these results. On the basis of these findings, hepatic hemangiosarcoma, with metastases to the lungs, omentum, subcutis, and bone, was diagnosed. This case study is the first report of spontaneous hepatic hemangiosarcoma in a cynomolgus macaque and the first case with metastasis to bone in a NHP.

An Interferon-Driven Oxysterol-Based Defense against Tumor-Derived Extracellular Vesicles.

Tumor-derived extracellular vesicles (TEV) "educate" healthy cells to promote metastases. We found that melanoma TEV downregulated type I interferon (IFN) receptor and expression of IFN-inducible cholesterol 25-hydroxylase (CH25H). CH25H produces 25-hydroxycholesterol, which inhibited TEV uptake. Low CH25H levels in leukocytes from melanoma patients correlated with poor prognosis. Mice incapable of downregulating the IFN receptor and Ch25h were resistant to TEV uptake, TEV-induced pre-metastatic niche, and melanoma lung metastases; however, ablation of Ch25h reversed these phenotypes. An anti-hypertensive drug, reserpine, suppressed TEV uptake and disrupted TEV-induced formation of the pre-metastatic niche and melanoma lung metastases. These results suggest the importance of CH25H in defense against education of normal cells by TEV and argue for the use of reserpine in adjuvant melanoma therapy.

Collagenoma in an African Clawed Frog (Xenopus laevis).

A 3-y-old female Xenopus laevis was reported for a gray mass on the abdomen. The frog was used for egg collection and was otherwise experimentally naïve. On physical exam, the frog was bright and active and had a firm, gray, lobulated mass (1.5 cm × 0.5 cm × 0.5 cm) in the cutaneous tissue of the left lateral abdomen. An excisional biopsy was performed under anesthesia, and the entire mass was removed and processed for histopathology. Microscopically, the dermis was greatly expanded by connective tissue with a marked decrease in the number of glands, and occasional degenerative glands were present. When stained with Masson trichrome, the excessive connective tissue stained blue, indicating that it was composed of collagen. With Verhoeff-van Gieson staining, the connective tissue stained bright red with an absence of black-staining material, demonstrating the presence of collagen and ruling out elastic fibers. In light of the morphology of the mass and the results of the special stains, the mass was diagnosed as a collagenoma. To our knowledge, this report is the first description of a collagenoma in X. laevis.

Neurocysticercosis in a Rhesus Macaque (Macaca mulatta).

An 8-y-old, intact, male rhesus macaque (Macaca mulatta) was sedated to undergo MRI in preparation for the implantation of cranial hardware. During imaging, 9 focal lesions were noted in the brain and musculature of the head. The lesions were hyperechoic with hypoechoic rims. The animal was deemed inappropriate for neuroscience research, and euthanasia was elected. Gross examination revealed multiple round, thick-walled, fluid-filled cysts (diameter, approximately 0.5 cm) in multiple tissues: one each in the left caudal lung lobe, left masseter muscle, and the dura overlying the brain and 8 throughout the gray and white matter of the brain parenchyma. Formalin-fixed sections of cyst-containing brain were stained with hematoxylin and eosin. Microscopic examination and molecular analysis of the COX1 (COI) gene recognized the causative organism as Taenia solium at 99.04% identity.

Evaluation of Presurgical Skin Preparation Agents in African Clawed Frogs (Xenopus laevis).

Despite the routine collection of oocytes from African clawed frogs (Xenopus laevis) for use in research, few studies have evaluated methods for preparing their skin for surgery. We evaluated 3 skin preparatory agents by examining their antibacterial efficacy and the gross and microscopic appearance of Xenopus skin after exposure. Frogs (n = 14) were sedated and treated (contact time, 10 min) with 0.9% sterile NaCl on one-half of the ventrum and with 0.5% povidone-iodine or 0.75% chlorhexidine on the other half. Bacterial cultures were obtained before and after skin treatment; bacteria were identified by mass spectrometry. To assess inflammation and degenerative changes, the incision sites were photographed and biopsied at 0, 1, and 7 d after surgery. We isolated at least 22 genera of bacteria from the skin of our frog population (mean ± SE, 5.21 ± 0.82 genera per frog). Iodine (2.00 ± 0.44 genera) and chlorhexidine (0.29 ± 0.76 genera) both had greater antimicrobial activity than did saline. Skin erythema did not correlate with treatment group. Histologic evidence of epidermal degeneration and necrosis was greater on days 1 and 7 after chlorhexidine treatment than after iodine or saline. In addition, frogs treated with chlorhexidine had a higher incidence of clinical illness associated with the exposure site. In summary, although chlorhexidine has adequate antimicrobial activity against organisms on X. laevis skin, it leads to skin damage and subsequent clinical complications. We therefore do not recommend chlorhexidine as a preoperative preparation agent in Xenopus.

Adverse effects of vapocoolant and topical anesthesia for tail biopsy of preweanling mice.

Tail biopsy of laboratory mice for genotyping purposes has been studied extensively to develop refinements for this common procedure. Our prior work assessed tail vertebral development in different mouse strains (age, 3 to 42 d) and analyzed behavior and activity in mice (age, 21 to 45 d) biopsied under isoflurane anesthesia. To assess the effects of biopsy on preweanling mice, we here evaluated BALB/cAnNCrl mice (n = 80; age, 18 to 21 d) that received topical vapocoolant (ethyl chloride), topical anesthetic (Cetacaine), or isoflurane anesthesia before undergoing a 5-mm or sham biopsy. Control mice did not receive any anesthetic intervention. Regardless of the anesthetic used, acute observation scores indicative of distress were increased at 10 min after biopsy, and locomotor activity was decreased, in biopsied compared with control mice. Acute observation scores at 10 min after biopsy were higher in mice that received ethyl chloride compared with isoflurane or no anesthesia. Microscopic analysis revealed that inflammatory changes in the distal tail remained elevated until 7 d after biopsy and were higher in tails exposed to ethyl chloride. Our findings indicate that vapocoolant, topical anesthesia, and inhaled isoflurane do not enhance the wellbeing of preweanling mice undergoing tail biopsy. Due to the lack of appreciable benefits and the presence of notable adverse effects, using vapocoolants or Cetacaine for this tail biopsy procedure in laboratory mice is unadvisable and we encourage the removal of these agents from institutional tail biopsy guidelines.

Deletion of the mitochondrial chaperone TRAP-1 uncovers global reprogramming of metabolic networks.

Reprogramming of metabolic pathways contributes to human disease, especially cancer, but the regulators of this process are unknown. Here, we have generated a mouse knockout for the mitochondrial chaperone TRAP-1, a regulator of bioenergetics in tumors. TRAP-1(-/-) mice are viable and showed reduced incidence of age-associated pathologies, including obesity, inflammatory tissue degeneration, dysplasia, and spontaneous tumor formation. This was accompanied by global upregulation of oxidative phosphorylation and glycolysis transcriptomes, causing deregulated mitochondrial respiration, oxidative stress, impaired cell proliferation, and a switch to glycolytic metabolism in vivo. These data identify TRAP-1 as a central regulator of mitochondrial bioenergetics, and this pathway could contribute to metabolic rewiring in tumors.

Simian immunodeficiency virus-infected macaques treated with highly active antiretroviral therapy have reduced central nervous system viral replication and inflammation but persistence of viral DNA.

BACKGROUND: During the era of highly active antiretroviral therapy (HAART), the prevalence of HIV-associated central nervous system (CNS) disease has increased despite suppression of plasma viremia. METHODS: In a simian immunodeficiency virus (SIV) model system in which all animals develop AIDS and 90% develop CNS disease by 3 months after inoculation, pigtailed macaques were treated with a regimen of tenofovir disoproxil fumarate, saquinavir, atazanavir, and an integrase inhibitor starting at 12 days after inoculation and were euthanized at approximately 175 days after inoculation. RESULTS: Plasma and cerebrospinal fluid (CSF) viral loads declined rapidly after the initiation of HAART. Brain viral RNA was undetectable at necropsy, but viral DNA levels were not different from those in untreated SIV-infected macaques. CNS inflammation was significantly reduced, with decreased brain expression of major histocompatibility complex class II and glial fibrillary acidic protein and reduced levels of CSF CCL2 and interleukin 6. Brain from treated macaques had significantly lower levels of interferon beta, type 1 interferon-inducible gene myxovirus (influenza) resistance A, and indolamine 2,3-dioxygenase messenger RNA, suggesting that immune hyperactivation was suppressed, and fewer CD4(+) and CD8(+) T cells, suggesting that trafficking of T cells from peripheral blood was reduced. Brain levels of CD68 protein and tumor necrosis factor alpha and interferon gamma RNA were reduced but were not significantly lower, indicating continued CNS inflammation. CONCLUSIONS: These data, generated in a rigorous, high-viral-load SIV-infected macaque model, showed that HAART provided benefits with respect to CNS viral replication and inflammation but that no change in the level of viral DNA and continued CNS inflammation occurred in some macaques.

HIV and SIV induce alterations in CNS CaMKII expression and activation: a potential mechanism for cognitive impairment.

The molecular mechanisms underlying learning and memory impairment in patients with HIV-associated neurological disease have remained unclear. Calcium/calmodulin-dependent kinase II (CaMKII) has key roles in synaptic potentiation and memory storage in neurons and also may have immunomodulatory functions. To determine whether HIV and simian immunodeficiency virus (SIV) induce alterations in CaMKII expression and/or activation (autophosphorylation) in the brain, we measured CaMKII alterations by quantitative immunoblotting in both an in vitro HIV/neuronal culture model and in vivo in an SIV-infected macaque model of HIV-associated neurological damage. Using primary rat hippocampal neuronal cultures treated with culture supernatants harvested from HIV-1-infected human monocyte-derived macrophages (HIV/MDM), we found that CaMKII activation declined after exposure of neurons to HIV/MDM. Consistent with our in vitro measurements, a significant decrease in CaMKII activation was present in both the hippocampus and frontal cortex of SIV-infected macaques compared with uninfected animals. In SIV-infected animals, total CaMKII expression in the hippocampus correlated well with levels of synaptophysin. Furthermore, CaMKII expression in both the hippocampus and frontal cortex was inversely correlated with viral load in the brain. These findings suggest that alterations in CaMKII may compromise synaptic function in the early phases of chronic neurodegenerative processes induced by HIV.

Minocycline attenuates HIV infection and reactivation by suppressing cellular activation in human CD4+ T cells.

Treatment of human immunodeficiency virus (HIV) infection with highly active antiretroviral therapy (HAART) is effective but can be associated with toxic effects and is expensive. Other options may be useful for long-term therapy. The immunomodulatory antibiotic minocycline could be an effective, low-cost adjunctive treatment to HAART. Minocycline mediated a dose-dependent decrease in single-cycle CXCR4-tropic HIV infection and decreased viral RNA after infection of CD4+ T cells with HIV NL4-3. Reactivation from latency was also decreased in a primary CD4+ T cell-derived model and in resting CD4+ T cells from HIV-infected patients. Minocycline treatment resulted in significant changes in activation marker expression and inhibited proliferation and cytokine secretion of CD4+ T cells in response to activation. This study demonstrates that minocycline reduces HIV replication and reactivation and decreases CD4+ T cell activation. The anti-HIV effects of minocycline are mediated by altering the cellular environment rather than directly targeting virus, placing minocycline in the class of anticellular anti-HIV drugs.

Coordinated regulation of SIV replication and immune responses in the CNS.

Central nervous system (CNS) invasion during acute-stage HIV-infection has been demonstrated in a small number of individuals, but there is no evidence of neurological impairment at this stage and virus infection in brain appears to be controlled until late-stage disease. Using our reproducible SIV macaque model to examine the earliest stages of infection in the CNS, we identified immune responses that differentially regulate inflammation and virus replication in the brain compared to the peripheral blood and lymphoid tissues. SIV replication in brain macrophages and in brain of SIV-infected macaques was detected at 4 days post-inoculation (p.i.). This was accompanied by upregulation of innate immune responses, including IFNbeta, IFNbeta-induced gene MxA mRNA, and TNFalpha. Additionally, IL-10, the chemokine CCL2, and activation markers in macrophages, endothelial cells, and astrocytes were all increased in the brain at four days p.i. We observed synchronous control of virus replication, cytokine mRNA levels and inflammatory markers (MHC Class II, CD68 and GFAP) by 14 days p.i.; however, control failure was followed by development of CNS lesions in the brain. SIV infection was accompanied by induction of the dominant-negative isoform of C/EBPbeta, which regulates SIV, CCL2, and IL6 transcription, as well as inflammatory responses in macrophages and astrocytes. This synchronous response in the CNS is in part due to the effect of the C/EBPbeta on virus replication and cytokine expression in macrophage-lineage cells in contrast to CD4+ lymphocytes in peripheral blood and lymphoid tissues. Thus, we have identified a crucial period in the brain when virus replication and inflammation are controlled. As in HIV-infected individuals, though, this control is not sustained in the brain. Our results suggest that intervention with antiretroviral drugs or anti-inflammatory therapeutics with CNS penetration would sustain early control. These studies further suggest that interventions should target HIV-infected individuals with increased CCL2 levels or HIV RNA in the CNS.