Assessment of dependence potential and abuse liability of Δ8-tetrahydrocannabinol in mice.

Delta-8-tetrahydrocannabinol (Δ8-THC) is a psychotropic cannabinoid produced in low quantities in the cannabis plant. Refinements in production techniques, paired with the availability of inexpensive cannabidiol substrate, have resulted in Δ8-THC being widely marketed as a quasi-legal, purportedly milder alternative to Δ9-THC. Yet, little research has probed the behavioral and physiological effects of repeated Δ8-THC use. The present study aimed to evaluate the effects of acute and repeated exposure to Δ8-THC. We hypothesized that Δ8-THC produces effects similar to Δ9-THC, including signs of drug tolerance and dependence. Adult male and female C57BL/6J mice were treated acutely with Δ8-THC (6.25-100 mg/kg, i.p.) or vehicle and tested in the tetrad battery to quantify cannabimimetic effects (i.e., catalepsy, antinociception, hypothermia, immobility) as compared with a non-selective synthetic cannabinoid (WIN 55,212-2) and Δ9-THC. As previously reported, Δ8-THC (≥12.5 mg/kg) induced cannabimimetic effects. Pretreatment with the CB1 receptor-selective antagonist rimonabant (3 mg/kg, i.p.) blocked each of these effects. In addition, repeated administration of Δ8-THC (50 mg/kg, s.c.) produced tolerance, as well as cross-tolerance to WIN 55,212-2 (10 mg/kg, s.c.) in tetrad, consistent with downregulated CB1 receptor function. Behavioral signs of physical dependence in the somatic signs, tail suspension, and marble burying assays were also observed following rimonabant-precipitated withdrawal from Δ8-THC (≥10 mg/kg BID for 6 days). Lastly, Δ8-THC produced Δ9-THC-like discriminative stimulus effects in both male and female mice. Together, these findings demonstrate that Δ8-THC produces qualitatively similar effects to Δ9-THC, including risk of drug dependence and abuse liability.

Large-N Rat Data Enables Phenotyping of Risky Decision-Making: A Retrospective Analysis of Brain Injury on the Rodent Gambling Task.

Decision-making is substantially altered after brain injuries. Patients and rats with brain injury are more likely to make suboptimal, and sometimes risky choices. Such changes in decision-making may arise from alterations in how sensitive individuals are to outcomes. To assess this, we compiled and harmonized a large dataset from four studies of TBI, each of which evaluated behavior on the Rodent Gambling Task (RGT). We then determined whether the following were altered: (1) sensitivity to overall contingencies, (2) sensitivity to immediate outcomes, or (3) general choice phenotypes. Overall sensitivity was evaluated using the matching law, immediate sensitivity by looking at the probability of switching choices given a win or loss, and choice phenotypes by k-means clustering. We found significant reductions in sensitivity to the overall outcomes and a bias toward riskier alternatives in TBI rats. However, the substantial individual variability led to poor overall fits in matching analyses. We also found that TBI caused a significant reduction in the tendency to repeatedly choose a given option, but no difference in win- or loss-specific sensitivity. Finally, clustering revealed 5 distinct decision-making phenotypes and TBI reduced membership in the "optimal" type. The current findings support a hypothesis that TBI reduces sensitivity to contingencies. However, in the case of tasks such as the RGT, this is not a simple shift to indiscriminate or less discriminate responding. Rather, TBI rats are more likely to develop suboptimal preferences and frequently switch choices. Treatments will have to consider how this behavior might be corrected.

Minocycline fails to treat chronic traumatic brain injury-induced impulsivity and attention deficits.

Traumatic brain injury (TBI) impacts millions worldwide and can cause lasting psychiatric symptoms. Chronic neuroinflammation is a characteristic of post-injury pathology and is also associated with psychiatric conditions such as ADHD and bipolar disorder. Therefore, the current study sought to determine whether TBI-induced impulsivity and inattention could be treated using minocycline, an antibiotic with anti-inflammatory properties. Rats were trained on the five-choice serial reaction time task (5CSRT), a measure of motor impulsivity and attention. After behavior was stable on the 5CSRT, rats received either a bilateral frontal TBI or sham procedure. Minocycline was given at either an early (1 h post-injury) or chronic (9 weeks post-injury) timepoint. Minocycline was delivered every 12 h for 5 days (45 mg/kg, i.p.). Behavioral testing on the 5CSRT began again after one week of recovery and continued for 12 more weeks, then rats were transcardially perfused. Impulsivity and inattention were both substantially increased following TBI. Minocycline had no therapeutic effects at either the early or late time points. TBI rats had increased lesion volume, but minocycline did not attenuate lesion size. Additionally, microglia count measured by IBA-1+ cells was only increased acutely after TBI, and minocycline did not differentially change the number of microglia in TBI rats. Despite this, minocycline had clear effects on the gut microbiome. Based on the results of this study, minocycline may have limited efficacy for post-injury psychiatric-like symptoms.

Melanocytic tumours of the nasal planum in cats: 10 cases (2004-2019).

OBJECTIVES: To better characterise signalment, biologic behaviour, and treatment outcome in melanocytic tumours of the nasal planum in cats. MATERIALS AND METHODS: Retrospective study in cats diagnosed with histopathologically confirmed melanocytic tumours on the nasal planum. RESULTS: Ten cats were identified with melanocytic tumours of the nasal planum. Pigmentation of the nasal planum seemed to be a predisposing factor. Seven cats were diagnosed histopathologically with a malignant melanoma, three with a benign melanocytoma. One of the cats developed a lymph node metastasis despite diagnosis of a well-differentiated melanocytic neoplasia. In four cases, a pigmented mass, which was initially stable over a long time, showed a sudden rapid progression suggesting malignant transformation. Treatments included hypofractionated radiation therapy (n = 6) and surgical resection (n = 1). In three cats no further treatment was pursued. Complete (n = 3) and partial (n = 3) remissions were observed in cats treated with radiation therapy; however, all experienced tumour progression or recurrence after a short period of time. Most of the cats (n = 7) had to be euthanased due to tumour progression (median survival time: 265 days). CLINICAL SIGNIFICANCE: The present case series provides insights in clinical presentation and clinical outcomes of cats with melanocytic tumours of the nasal planum.

WNT3A and the induction of the osteogenic differentiation in adipose tissue derived mesenchymal stem cells.

Adipose tissue derived stem cells (ASCs) can easily be isolated, but the osteogenic differentiation potential is limited. To improve this differentiation potential, more investigations are required about signaling proteins for the induction of the osteogenic differentiation. This study focused on the WNT3A protein, because little is known about the canonical WNT signaling pathway and the osteogenic differentiation of ASCs. The alkaline phosphatase (ALP) activity was measured for the evaluation of the osteogenic differentiation. WNT3A and Dickkopf-related protein 1 (DKK1) were used for the activation and the inhibition of the canonical WNT signaling pathway, respectively. For control we manipulated the bone morphogenetic protein (BMP) pathway in ASCs with BMP2 and NOGGIN (BMP pathway inhibitor). WNT3A stimulated significantly the ALP activity in ASCs, while BMP2, DKK1 and NOGGIN did not induce highly the ALP activity in ASCs. Moreover, an osteogenic differentiation medium with dexamethasone and WNT3A increased the ALP activity, but the gene expression of osteoblast markers and the biomineralization after long-term cultures were not increased. In contrast, ASCs differentiated into adipocyte-like cells in all tested differentiation media. WNT3A did not repress the expression of the adipogenic transcription factor Peroxisome Proliferator-Activated Receptor Gamma (PPARG). In conclusion, WNT3A supports early stages such as the ALP activity, but it does neither improve later stages of the osteogenic differentiation nor it inhibits the genuine adipogenic differentiation of ASCs.

The hedgehog-signaling pathway is repressed during the osteogenic differentiation of dental follicle cells.

Dental follicle stem cells (DFCs) are precursor cells of alveolar osteoblasts, and previous studies have shown that the growth factor bone morphogenetic protein (BMP)2 induces the osteogenic differentiation of DFCs. However, the molecular mechanism down-stream of the induction of the osteogenic differentiation by BMP2 remains elusive. We investigated therefore the phosphoproteome of DFCs after the induction of the osteogenic differentiation with BMP2. In this study, phosphoproteins of the hedgehog "off" state were differentially expressed. Further analyses revealed that BMP2 induced the expression of repressors of the hedgehog-signaling pathway such as Patched 1 (PTCH1), Suppressor of Fused (SUFU), and Parathyroid Hormone-Related Peptide (PTHrP). Previous studies suggested that hedgehog proteins induce the osteogenic differentiation of mesenchymal stem cells via a paracrine pathway. Indian hedgehog (IHH) induced the expression of the osteogenic transcription factor RUNX2. However, a supplementation of the BMP2-based osteogenic differentiation medium with IHH did not induce the expression of RUNX2. Moreover, IHH inhibited slightly the ALP activity and the mineralization of osteogenic-differentiated DFCs. In conclusion, our results suggest that BMP2 inhibits the hedgehog signaling after the induction of the osteogenic differentiation in DFCs.

The extracellular concentration of osteocalcin decreased in dental follicle cell cultures during biomineralization.

The secretion of osteocalcin (OCN) is an excellent differentiation marker for the osteogenic differentiation. This study investigated the secretion of OCN during the osteogenic differentiation of DFCs. During the differentiation of DFCs the extracellular concentrations of OCN were higher in standard cell culture medium than in osteogenic differentiation medium. However, after 4 weeks in the osteogenic differentiation medium the extracellular OCN concentration decreased strongly, whereas the concentration remains high in the control medium. At this point in time DFCs formed connective tissue like structures with mineralized clusters and OCN. Real-time RT-PCR analyses and western-blot analyses proved that OCN was expressed in both cell culture media. However, the expression of the mRNA was inhibited in the osteogenic differentiation medium. These results suggest that DFCs secrete constitutively OCN into the cell culture medium and that the osteogenic differentiation medium suppresses the gene expression of OCN. Moreover, OCN imbeds into the extracellular matrix after the formation of connective tissue like structures, and the soluble OCN in the cell culture medium disappears. Hence, extracellular OCN in the cell culture medium is not a marker for the osteogenic differentiation of DFCs.

The parathyroid hormone-related protein is secreted during the osteogenic differentiation of human dental follicle cells and inhibits the alkaline phosphatase activity and the expression of DLX3.

The dental follicle is involved in tooth eruption and it expresses a great amount of the parathyroid hormone-related protein (PTHrP). PTHrP as an extracellular protein is required for a multitude of different regulations of enchondral bone development and differentiation of bone precursor cells and of the development of craniofacial tissues. The dental follicle contains also precursor cells (DFCs) of the periodontium. Isolated DFCs differentiate into periodontal ligament cells, alveolar osteoblast and cementoblasts. However, the role of PTHrP during the human periodontal development remains elusive. Our study evaluated the influence of PTHrP on the osteogenic differentiation of DFCs under in vitro conditions for the first time. The PTHrP protein was highly secreted after 4days of the induction of the osteogenic differentiation of DFCs with dexamethasone (2160.5pg/ml±345.7SD. in osteogenic differentiation medium vs. 315.7pg/ml±156.2SD. in standard cell culture medium; Student's t Test: p<0.05 (n=3)). We showed that the supplementation of the osteogenic differentiation medium with PTHrP inhibited the alkaline phosphatase activity and the expression of the transcription factor DLX3, but the depletion of PTHrP did not support the differentiation of DFCs. Previous studies have shown that Indian Hedgehog (IHH) induces PTHrP and that PTHrP, in turn, inhibits IHH via a negative feedback loop. We showed that SUFU (Suppressor Of Fused Homolog) was not regulated during the osteogenic differentiation in DFCs. So, neither the hedgehog signaling pathway induced PTHrP nor PTHrP suppressed the hedgehog signaling pathway during the osteogenic differentiation in DFCs. In conclusion, our results suggest that PTHrP regulates independently of the hedgehog signaling pathway the osteogenic differentiated in DFCs.

Cancer stem cell-like cells from a single cell of oral squamous carcinoma cell lines.

Resistance of oral squamous cell carcinomas (OSCC) to conventional chemotherapy or radiation therapy might be due to cancer stem cells (CSCs). The development of novel anticancer drugs requires a simple method for the enrichment of CSCs. CSCs can be enriched from OSCC cell lines, for example, after cultivation in serum-free cell culture medium (SFM). In our study, we analyzed four OSCC cell lines for the presence of CSCs. CSC-like cells could not be enriched with SFM. However, cell lines obtained from holoclone colonies showed CSC-like properties such as a reduced rate of cell proliferation and a reduced sensitivity to Paclitaxel in comparison to cells from the parental lineage. Moreover, these cell lines differentially expressed the CSC-marker CD133, which is also upregulated in OSCC tissues. Interestingly, CD133(+) cells in OSCC tissues expressed little to no Ki67, the cell proliferation marker that also indicates reduced drug sensitivity. Our study shows a method for the isolation of CSC-like cell lines from OSCC cell lines. These CSC-like cell lines could be new targets for the development of anticancer drugs under in vitro conditions.

[Women caring for women: a study at the "Viva Maria" shelter, Porto Alegre, Rio Grande do Sul, Brazil]. / Mulheres cuidando de mulheres: um estudo sobre a Casa de Apoio Viva Maria, Porto Alegre, Rio Grande do Sul, Brasil.

This research was conducted in Porto Alegre, Rio Grande do Sul, with a sample of battered women selected from a government shelter called the "Casa Viva Maria". We analyzed data on 110 women staying at the shelter during the previous two years (January 1996-June 1998). The profile of the women was as follows: abused women were young (mean age 29 years), all had low socioeconomic status, 12% were illiterate, 21% were black, 80% reported frequent abuse by their partners, and 18% had returned to violent homes. The researchers visited 34 former lodgers from the shelter and invited them to participate in a series of evaluation workshops. A total of 118 persons, including mothers and children, attended three evaluation meetings. During this process, researchers encouraged participants to express opinions, perceptions, and feelings about their past experience in the shelter and their own concept of violence. Finally, a focal group was organized with the "Viva Maria" staff members. Female workers reported how their job had been helpful for their personal development and had helped change their own lives.

Polypoidal choroidal vasculopathy in exudative and haemorrhagic pigment epithelial detachments.

AIMS: To determine the prevalence of polypoidal choroidopathy in consecutive patients presenting with large haemorrhagic and exudative neurosensory retinal and retinal pigment epithelial detachments (PEDs) of over 2 mm in diameter in the absence of drusen. METHODS: 40 patients were identified over a 5 month period of which 29 had haemorrhagic detachments, and 11 had purely exudative detachments. All had indocyanine green (ICG) angiography, and the presence was sought of large blood vessels in the choroid associated with localised dilated terminals that filled slowly and leaked ICG. RESULTS: In 34 cases (85%) there was an appearance consistent with previous descriptions of idiopathic polypoidal choroidal vasculopathy. Of the six without polypoidal lesions the disorder was attributed to choroidal neovascularisation in four, chorioretinitis in one, and a fibrovascular PED in one. Of those with polypoidal lesions 20 (65%) were female, the mean age was 65.4 years (range 44-88), and 25 (74%) were white, seven (20%) black, and two (6%) east Asian. Eight had a history of hypertension. Visual acuity varied from 6/6 to counting fingers in the involved eye (mean 6/24). Bilateral polypoidal choroidal lesions were demonstrated in 16 patients (47%). The predominant location for these lesions was the macular region in 23 patients (68%). Polypoidal vasculopathy was found in 16 patients (47%) who had a previous diagnosis of age related macular disease (AMD). No patients had evidence of intraocular inflammation. CONCLUSIONS: In a largely white patient population a high proportion of patients with haemorrhagic and exudative PEDs has evidence of polypoidal lesions on ICG angiography.