Taste-immune associative learning amplifies immunopharmacological effects and attenuates disease progression in a rat glioblastoma model.

Mechanistic target of rapamycin (mTOR)-signaling is one key driver of glioblastoma (GBM), facilitating tumor growth by promoting the shift to an anti-inflammatory, pro-cancerogenic microenvironment. Even though mTOR inhibitors such as rapamycin (RAPA) have been shown to interfere with GBM disease progression, frequently chaperoned toxic drug side effects urge the need for developing alternative or supportive treatment strategies. Importantly, previous work document that taste-immune associative learning with RAPA may be utilized to induce learned pharmacological placebo responses in the immune system. Against this background, the current study aimed at investigating the potential efficacy of a taste-immune associative learning protocol with RAPA in a syngeneic GBM rat model. Following repeated pairings of a novel gustatory stimulus with injections of RAPA, learned immune-pharmacological effects could be retrieved in GBM-bearing animals when re-exposed to the gustatory stimulus together with administering 10 % amount of the initial drug dose (0.5 mg/kg). These inhibitory effects on tumor growth were accompanied by an up-regulation of central and peripheral pro-inflammatory markers, suggesting that taste-immune associative learning with RAPA promoted the development of a pro-inflammatory anti-tumor microenvironment that attenuated GBM tumor growth to an almost identical outcome as obtained after 100 % (5 mg/kg) RAPA treatment. Together, our results confirm the applicability of taste-immune associative learning with RAPA in animal disease models where mTOR overactivation is one key driver. This proof-of-concept study may also be taken as a role model for implementing learning protocols as alternative or supportive treatment strategy in clinical settings, allowing the reduction of required drug doses and side effects without losing treatment efficacy.

Glioma: molecular signature and crossroads with tumor microenvironment.

In patients with glioblastoma, the average survival time with current treatments is short, mainly due to recurrences and resistance to therapy. This insufficient treatment success is, in large parts, due to the tremendous molecular heterogeneity of gliomas, which affects the overall prognosis and response to therapies and plays a vital role in gliomas' grading. In addition, the tumor microenvironment is a major player for glioma development and resistance to therapy. Active communication between glioma cells and local or neighboring healthy cells and the immune environment promotes the cancerogenic processes and contributes to establishing glioma stem cells, which drives therapy resistance. Besides genetic alterations in the primary tumor, tumor-released factors, cytokines, proteins, extracellular vesicles, and environmental influences like hypoxia provide tumor cells the ability to evade host tumor surveillance machinery and promote disease progression. Moreover, there is increasing evidence that these players affect the molecular biological properties of gliomas and enable inter-cell communication that supports pro-cancerogenic cell properties. Identifying and characterizing these complex mechanisms are inevitably necessary to adapt therapeutic strategies and to develop novel measures. Here we provide an update about these junctions where constant traffic of biomolecules adds complexity in the management of glioblastoma.

Rodent Models to Analyze the Glioma Microenvironment.

Animal models are still indispensable for understanding the basic principles of glioma development and invasion. Preclinical approaches aim to analyze the treatment efficacy of new drugs before translation into clinical trials is possible. Various animal disease models are available, but not every approach is useful for addressing specific questions. In recent years, it has become increasingly evident that the tumor microenvironment plays a key role in the nature of glioma. In addition to providing an overview, this review evaluates available rodent models in terms of usability for research on the glioma microenvironment.

Syphilitic Gummata in the Central Nervous System: A Narrative Review and Case Report about a Noteworthy Clinical Manifestation.

Infection with Treponema pallidum is on the rise. In this narrative literature review, we show that the incidence of rare manifestations of syphilis, such as intracerebral gummata, is increasing and should be considered in the differential diagnosis of intracerebral lesions. With the exemplary case that we present here, we aim to raise awareness of the resurgence of this disease, which should be considered in the differential diagnosis of intracerebral lesions, especially for patients who have a risk profile for syphilis, and serological testing for T. pallidum prior to surgery should be discussed in order to avoid an unnecessary operation.

Incomplete reminder cues trigger memory reconsolidation and sustain learned immune responses.

Peripheral immune responses can be modulated by taste-immune associative learning where the presentation of a sweet taste as conditioned stimulus (CS) is paired with the injection of an immunosuppressive substance as unconditioned stimulus (US). Previous findings demonstrate conditioned immunopharmacological properties of the mechanistic target of rapamycin (mTOR)-inhibitor rapamycin, a drug used to ameliorate neurological diseases and for the prevention of graft rejection. However, conditioned responses gradually weaken over time and eventually disappear following repeated exposure to the CS in the absence of the US. Thus, in order to employ learning paradigms in clinical conditions as supportive immunopharmacological therapy it is important to understand the central and peripheral mechanisms of how learned immune responses can be protected from extinction. Against this background, the present study used a taste-immune learning paradigm with rapamycin as US (5 mg/kg). By applying only 10% (0.5 mg/kg) of the therapeutic dose rapamycin together with the CS (taste stimulus) during eight retrieval trials, conditioned animals still displayed suppressed interleukin-10 production and T cell proliferation in splenocytes as well as diminished activity of the mTOR target protein p70s6k in amygdala tissue samples. Together, these findings indicate that reminder cues in form of only 10% (0.5 mg/kg) of the therapeutic dose rapamycin together with the CS (taste stimulus) at retrieval preserved the memory of conditioned properties of rapamycin, characterizing this approach as a potential supportive tool in peripheral and central pharmacotherapy with the aim to maximize the therapeutic outcome for the patient's benefit.

A step-by-step guide for microsurgical collection of uncontaminated cerebrospinal fluid from rat cisterna magna.

BACKGROUND: Cerebrospinal fluid (CSF) analysis is of significant clinical importance for the diagnosis of diseases. In humans, CSF is easily accessible and can be collected using minimally invasive methods. However, obtaining uncontaminated CSF from rats is still challenging. NEW METHOD: This study described a microsurgical technique for sampling large quantities (>200 µL) of clear and non-blood-contaminated CSF from the rat cisterna magna in a comprehensible step-by-step guide and provided a graphical visualization. RESULTS: CSF was sampled in 5-10 min (n = 29 animals; average surgical time 7.6 min). In visual control, 28 samples (97 %) of clear and uncontaminated CSF were obtained. The volume of CSF collected was 124-337 µL, with an average volume of 207 µL/sample. Using the Valsalva maneuver, we could collect higher volumes (up to 400 µL) several times. COMPARISON WITH EXISTING METHOD(S): There is no need for special surgical skills to perform this method accurately. The method takes a few minutes longer than a percutaneous puncture (<1 min in pups). However, the volume of CSF obtained using the percutaneous approach in adult rats (50-70 µL) is comparatively low. CONCLUSIONS: We described a practical method of sampling CSF from rats that enables large volumes of CSF to be collected without blood contamination. No special surgical skills are required to use this method. With proper practice, the time between skin incision and CSF sampling is <10 min. Depending on the experimental design requirements, some additional time must be planned for wound closure.

Neurobehavioral effects in rats with experimentally induced glioblastoma after treatment with the mTOR-inhibitor rapamycin.

Psychiatric symptoms as seen in affective and anxiety disorders frequently appear during glioblastoma (GBM) treatment and disease progression, additionally deteriorate patient's daily life routine. These central comorbidities are difficult to recognize and the causes for these effects are unknown. Since overactivation of mechanistic target of rapamycin (mTOR)- signaling is one key driver in GBM growth, the present study aimed at examining in rats with experimentally induced GBM, neurobehavioral consequences during disease progression and therapy. Male Fisher 344 rats were implanted with syngeneic RG2 tumor cells in the right striatum and treated with the mTOR inhibitor rapamycin (3 mg/kg; once daily, for eight days) before behavioral performance, brain protein expression, and blood samples were analyzed. We could show that treatment with rapamycin diminished GBM tumor growth, confirming mTOR-signaling as one key driver for tumor growth. Importantly, in GBM animals' anxiety-like behavior was observed but only after treatment with rapamycin. These behavioral alterations were moreover accompanied by aberrant glucocorticoid receptor, phosphorylated p70 ribosomal S6 kinase alpha (p-p70s6k), and brain derived neurotrophic factor protein expression in the hippocampus and amygdala in the non-tumor-infiltrated hemisphere of the brain. Despite the beneficial effects on GBM tumor growth, our findings indicate that therapy with rapamycin impaired neurobehavioral functioning. This experimental approach has a high translational value. For one, it emphasizes aberrant mTOR functioning as a central feature mechanistically linking complex brain diseases and behavioral disturbances. For another, it highlights the importance of elaborating the cause of unwanted central effects of immunosuppressive and antiproliferative drugs used in transplantation medicine, immunotherapy, and oncology.

Surgery for Degenerative Cervical Myelopathy: What Really Counts?

STUDY DESIGN: Retrospective study (data analysis). OBJECTIVE: The purpose of this study was to assess the role of different factors on postoperative outcome of patients with degenerative cervical myelopathy (DCM). SUMMARY OF BACKGROUND DATA: Ongoing degenerative changes of DCM lead to progressive neurological deficits. The optimal timing of surgical treatment is still unclear, especially in patients with mild DCM. METHODS: Patients with DCM treated in our clinic between 2007 and 2016 were retrospectively analyzed. Pre- and postoperative neurological function was assessed by the modified Japanese Orthopaedic Association Score (mJOA Score) at different stages. The minimum clinically important difference (MCID) was used to evaluate the improvement after surgery. The comorbidities were recorded using the Charlson Comorbidity Index (CCI). Possible associations between age, sex, CCI, preoperative symptoms duration, high signal intensity (SI) on T2-weighted magnetic resonance imaging (MRI) with mJOA Score and MCID were analyzed using univariate analysis and multivariate regression models. Additionally, subgroup analysis was performed according to the severity of DCM (mild: mJOA Score ≥15 points; moderate: mJOA Score of 12-14 points; and severe: mJOA Score <12 points). RESULTS: The mean age of the final cohort (n = 411) was 62.6 years (range: 31-96 years), 36.0% were females. High SI on T2-weighted MRI was detected in 60.3% of the cases. In the multivariate analysis, patients' age (P = 0.005), higher CCI (P = 0.001), and presence of high SI on T2-weighted MRI (P = 0.0005) were associated independently with lower pre- and postoperative mJOA Score and postoperative MCID. Subgroup analysis revealed age and high SI on T2-weighted MRI as predictors of pre- and postoperative mJOA. However, symptom duration did not influence neurological outcome according to the severity of DCM. CONCLUSION: Surgery for DCM leads to significant functional improvement. However, better outcome was observed in younger individuals with lower CCI and absence of radiographic myelopathy signs. Therefore, DCM surgery, particularly before occurrence of high SI on MRI, seems to be essential for postoperative functional improvement regardless the above-mentioned confounders.Level of Evidence: 3.

Dose-Dependent Acute Effects of Everolimus Administration on Immunological, Neuroendocrine and Psychological Parameters in Healthy Men.

The rapamycin analogue everolimus (EVR) is a potent inhibitor of the mammalian target of rapamycin (mTOR) and clinically used to prevent allograft rejections as well as tumor growth. The pharmacokinetic and immunosuppressive efficacy of EVR have been extensively reported in patient populations and in vitro studies. However, dose-dependent ex vivo effects upon acute EVR administration in healthy volunteers are rare. Moreover, immunosuppressive drugs are associated with neuroendocrine changes and psychological disturbances. It is largely unknown so far whether and to what extend EVR affects neuroendocrine functions, mood, and anxiety in healthy individuals. Thus, in the present study, we analyzed the effects of three different clinically applied EVR doses (1.5, 2.25, and 3 mg) orally administered 4 times in a 12-hour cycle to healthy male volunteers on immunological, neuroendocrine, and psychological parameters. We observed that oral intake of medium (2.25 mg) and high doses (3 mg) of EVR efficiently suppressed T cell proliferation as well as IL-10 cytokine production in ex vivo mitogen-stimulated peripheral blood mononuclear cell. Further, acute low (1.5 mg) and medium (2.25 mg) EVR administration increased state anxiety levels accompanied by significantly elevated noradrenaline (NA) concentrations. In contrast, high-dose EVR significantly reduced plasma and saliva cortisol as well as NA levels and perceived state anxiety. Hence, these data confirm the acute immunosuppressive effects of the mTOR inhibitor EVR and provide evidence for EVR-induced alterations in neuroendocrine parameters and behavior under physiological conditions in healthy volunteers.

Superiority of preventive antibiotic treatment compared with standard treatment of poststroke pneumonia in experimental stroke: a bed to bench approach.

Stroke patients are prone to life-threatening bacterial pneumonia. Previous experimental stroke studies have demonstrated that preventive antibiotic treatment (PAT) improves outcome compared with placebo treatment, which however does not model the clinical setting properly. Here we investigate whether PAT is superior to the current clinical 'gold standard' for treating poststroke infections. Therefore, we modeled stroke care according to the current stroke guidelines recommending early antibiotic treatment after diagnosing infections. To reliably diagnose pneumonia in living mice, we established a general health score and a magnetic resonance imaging protocol for radiologic confirmation. Compared with standard treatment after diagnosis by these methods, PAT not only abolished pneumonia successfully but also improved general medical outcome. Both, preventive and standard antibiotic treatment using enrofloxacin improved survival in a similar way compared with placebo treatment. However, in contrast to standard treatment, only PAT improved functional outcome assessed by gait analysis. In conclusion, standard and preventive treatment approach reduced poststroke mortality, however at the cost of a worse neurologic outcome compared with preventive approach. These data support the concept of PAT for treating patients at risk for poststroke infections and warrant phase III trials to prove this concept in clinical setting.

Gait analysis as a method for assessing neurological outcome in a mouse model of stroke.

Ameliorating stroke induced neurological deficits is one of the most important goals of stroke therapy. In order to improve stroke outcome, novel treatment approaches as well as animal stroke models predictive for the clinical setting are of urgent need. One of the main obstacles in experimental stroke research is measuring long-term outcome, in particular in mouse models of stroke. On the other hand, assessing functional deficits in animal models of stroke is critical to improve the prediction of preclinical findings. Automated gait analysis provides a sensitive tool to examine locomotion and limb coordination in small rodents. Comparing mice before and 10 days after experimental stroke (60 min MCAo) we observed a significant decrease in maximum contact area, stride length and swing speed in the hind limbs, especially the contralateral one. Mice showed a disturbed interlimb coordination represented by changes in regularity index and phase dispersion. To assess whether gait analysis is applicable to assess improvements by neuroprotective compounds, we applied a model calculation and approached common statistical problems. In conclusion, gait analysis is a promising tool to assess mid- to long-term outcome in experimental stroke research.