Antti Nurmi

Pyrrolidine dithiocarbamate inhibits translocation of nuclear factor kappa-B in neurons and protects against brain ischaemia with a wide therapeutic time window

Pyrrolidine dithiocarbamate (PDTC) is an antioxidant and inhibitor of transcription factor nuclear factor kappa‐B (NF‐κB). Because the role of NF‐κB in brain injury is controversial and another NF‐κB inhibiting thiocarbamate, DDTC, was recently shown to increase ischaemic brain damage, we investigated the effect of PDTC on transient brain ischaemia. Ischaemia was induced by occlusion of the middle cerebral artery (MCAO). In Wistar rats, the PDTC treatment started even 6 h after MCAO reduced the infarction volume by 48%. PDTC protected against MCAO also in spontaneously hypertensive rats and against forebrain ischaemia in Mongolian gerbils. PDTC prevented NF‐κB activation in the ischaemic brain as determined by reduced DNA binding and nuclear translocation of NF‐κB in neurons. PDTC had anti‐inflammatory effect by preventing induction of NF‐κB‐regulated pro‐inflammatory genes. In ischaemic rats, NF‐κB was localized in cyclo‐oxygenase‐2‐immunoreactive neurons. Blood cytokine levels were not altered by ischaemia or PDTC. When cultured neurons were exposed to an excitotoxin, no production of reactive oxygen species was detected, but PDTC provided protection and prevented nuclear translocation of NF‐κB. The clinically approved PDTC and its analogues may act as anti‐inflammatories and may be safe therapies in stroke with a wide time window.

Cerebral Blood Volume Alterations in the Perilesional Areas in the Rat Brain after Traumatic Brain Injury—Comparison with Behavioral Outcome

In the traumatic brain injury (TBI) the initial impact causes both primary injury, and launches secondary injury cascades. One consequence, and a factor that may contribute to these secondary changes and functional outcome, is altered hemodynamics. The relative cerebral blood volume (CBV) changes in rat brain after severe controlled cortical impact injury were characterized to assess their interrelations with motor function impairment. Magnetic resonance imaging (MRI) was performed 1, 2, 4 h, and 1, 2, 3, 4, 7, and 14 days after TBI to quantify CBV and water diffusion. Neuroscore test was conducted before, and 2, 7, and 14 days after the TBI. We found distinct temporal profile of CBV in the perilesional area, hippocampus, and in the primary lesion. In all regions, the first response was drop of CBV. Perifocal CBV was reduced for over 4 days thereafter gradually recovering. After the initial drop, the hippocampal CBV was increased for 2 weeks. Neuroscore demonstrated severely impaired motor functions 2 days after injury (33% decrease), which then slowly recovered in 2 weeks. This recovery parallelled the recovery of perifocal CBV. CBV MRI can detect cerebrovascular pathophysiology after TBI in the vulnerable perilesional area, which seems to potentially associate with time course of sensory-motor deficit.

Deficiency of prolyl oligopeptidase in mice disturbs synaptic plasticity and reduces anxiety-like behaviour, body weight, and brain volume.

Prolyl oligopeptidase (PREP) has been implicated in neurodegeneration and neuroinflammation and has been considered a drug target to enhance memory in dementia. However, the true physiological role of PREP is not yet understood. In this paper, we report the phenotyping of a mouse line where the PREP gene has been knocked out. This work indicates that the lack of PREP in mice causes reduced anxiety but also hyperactivity. The cortical volumes of PREP knockout mice were smaller than those of wild type littermates. Additionally, we found increased expression of diazepam binding inhibitor protein in the cortex and of the somatostatin receptor-2 in the hippocampus of PREP knockout mice. Furthermore, immunohistochemistry and tail suspension test revealed lack of response of PREP knockout mice to lipopolysaccharide insult. Further analysis revealed significantly increased levels of polysialylated-neural cell adhesion molecule in PREP deficient mice. These findings might be explained as possible alteration in brain plasticity caused by PREP deficiency, which in turn affect behaviour and brain development.

Alteration of prolyl oligopeptidase and activated α-2-macroglobulin in multiple sclerosis subtypes and in the clinically isolated syndrome

Prolyl oligopeptidase (PREP) has been considered as a drug target for the treatment of neurodegenerative diseases. In plasma, PREP has been found altered in several disorders of the central nervous system including multiple sclerosis (MS). Oxidative stress and the levels of an endogenous plasma PREP inhibitor have been proposed to decrease PREP activity in MS. In this work, we measured the circulating levels of PREP in patients suffering of relapsing remitting (RR), secondary progressive (SP), primary progressive (PP) MS, and in subjects with clinically isolated syndrome (CIS). We found a significantly lower PREP activity in plasma of RRMS as well as in PPMS patients and a trend to reduced activity in subjects diagnosed with CIS, compared to controls. No signs of oxidative inactivation of PREP, and no correlation with the endogenous PREP inhibitor, identified as activated α-2-macroglobulin (α2M*), were observed in any of the patients studied. However, a significant decrease of α2M* was recorded in MS. In cell cultures, we found that PREP specifically stimulates immune active cells possibly by modifying the levels of fibrinogen β, thymosin β4, and collagen. Our results open new lines of research on the role of PREP and α2M* in MS, aiming to relate them to the diagnosis and prognosis of this devastating disease.

Neuroprotective properties of the non-peptidyl radical scavenger IAC in rats following transient focal cerebral ischemia

Experimental evidence suggests that reactive free radicals are generated during brain ischemia. We investigated the effect of a novel brain penetrant, low molecular weight, non-peptidyl carbon, oxygen- and nitrogen-centered radical scavenger, IAC, on infarct volume and sensory-motor performance in a rat transient middle cerebral artery occlusion model (tMCAO). Rats received 90 min tMCAO and treated with i.p. or i.v. injections of vehicle or IAC following tMCAO. Sensory-motor performance was evaluated by neuroscore tests (NS). Cerebral infarct volume was evaluated at 72 h after tMCAO. Rats treated with IAC i.p. (1 or 6 h after the onset of tMCAO) or i.v. (1 h after the onset of tMCAO) showed significant improvement in NS during the 3 or 21 day follow-up period when compared to vehicle treated rats. Cerebral infarct volumes were significantly decreased compared to vehicle in rats receiving IAC i.p. 1 h or 6 h after occlusion, approximately 30.5% decrease compared to vehicle, or i.v. 1 h after the onset of tMCAO, 48.6% decrease compared to vehicle. These results demonstrate that IAC has neuroprotective properties with a wide therapeutic window following tMCAO in rats. IAC could therefore be a candidate for the treatment of stroke.

The radical scavenger IAC (bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl) decantionate) decreases mortality, enhances cognitive functions in water maze and reduces amyloid plaque burden in hAβPP transgenic mice.

The purpose of this study was to evaluate the efficacy of the radical scavenger IAC (bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl) decantionate) in alleviating behavioral deficits and reducing amyloid-β (Aβ) accumulation in an Alzheimers disease (AD) transgenic Tg2576 mouse model. Daily treatment with IAC (3-30 mg/kg, i.p.) was started at the age of 6 months and continued until the mice were 13 months old. At the age of 9 months and again at 12 months, the mice were tested in open field and water maze tests. At the age of 13 months, the mice were sacrificed and the brains processed for immunohistochemistry. Mortality was significantly reduced in all IAC-treated groups. In addition, IAC treatment improved the water maze hidden platform training performance but had no effect on motor activity in the open field or water maze swim speed in transgenic mice. Lastly, IAC treatment (10 mg/kg) significantly reduced the cortical Aβ plaque burden. In vitro, IAC is able to increase the number of neurites and neurite branches in cultured cortical primary neurons. In conclusion, IAC slowed down the development of the AD-like phenotype in Tg2576 mice and accelerated neurite growth in cultured neurons.

Inaugural Charles River World Congress on Animal Models in Drug Discovery and Development

© The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. A1 Bench to bedside in oncology: translation of cancer vaccines from mouse models to human clinical trials Jay A. Berzofsky Vaccine Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 USA Correspondence: Jay A. Berzofsky (berzofsj@mail.nih.gov) Journal of Translational Medicine 2017, 15(Suppl 3): A1

Lysophosphatidyl Choline Induced Demyelination in Rat Probed by Relaxation along a Fictitious Field in High Rank Rotating Frame

In this work a new MRI modality entitled Relaxation Along a Fictitious Field in the rotating frame of rank 4 (RAFF4) was evaluated in its ability to detect lower myelin content in lysophosphatidyl choline (LPC)-induced demyelinating lesions. The lesions were induced in two areas of the rat brain with either uniform or complex fiber orientations, i.e., in the corpus callosum (cc) and dorsal tegmental tract (dtg), respectively. RAFF4 showed excellent ability to detect demyelinated lesions and good correlation with myelin content in both brain areas. In comparison, diffusion tensor imaging metrices, fractional anisotropy, mean diffusivity and axonal and radial diffusivity, and magnetization transfer (MT) metrices, longitudinal relaxation during off-resonance irradiation and MT ratio, either failed to detect demyelination in dtg or showed lower correlation with myelin density quantified from gold chloride stained histological sections. Good specifity of RAFF4 to myelin was confirmed by its low correlation with cell density assesed from Nissl stained sections as well as its lack of sensitivity to pH changes in the physiological range as tested in heat denaturated bovine serum albumin phantoms. The excellent ability of RAFF4 to detect myelin content and its insensitivity to fiber orientation distribution, gliosis and pH, together with low specific absorption rate, demonstrates the promise of rotating frame of rank n (RAFFn) as a valuable MRI technique for non-invasive imaging of demyelinating lesions.

ASSESSMENT OF LEARNING AND MEMORY IN THE CVN MOUSE MODEL OF ALZHEIMER’S DISEASE IN AUTOMATED TOUCH SCREEN CHAMBERS

Background: According to previously published data and internal model validation, the CVN mouse model of AD exhibits cognitive impairments in contextual fear conditioning, Barnes maze and radial arm water maze most prominently at 9–12 months of age. To determine if impaired learning can be detected at an earlier age, we evaluated their cognitive performance by using a touch screen operant platform (Horner et al., 2013), which is similar to computerized CANTAB testing widely used in clinical setting. Methods: Cohorts of 4–5-month old male CVN (APPSDI/NOS2 knockout) and associated wild type (WT) mice were obtained from Charles River (Sulzfeld, Germany). Following acclimatization, animals were introduced to a restricted diet throughout the study period. Testing was conducted in Bussey-Saksida mouse touch screen chambers (Campden Instruments, Loughborough, UK). Mice were trained to develop screen-touching behavior using a 5-stage pretraining algorithm. Thereafter, they were tested in their ability to visually discriminate alternative black and white “linesgrid” stimuli on the touch screen. In order to test cognitive flexibility, the task of reversal of the acquired visual discrimination was implemented by switching the contingencies used in the visual discrimination test. Results:During pretraining, CVN mice and WT mice required similar number of sessions to achieve the pretraining criterion. Furthermore, the majority of mice successfully acquired visual discrimination within 20 days, again without significant differences in the number of sessions required to reach the discrimination criterion between CVN and WT mice. Conclusions:We found that CVN mice did not exhibit gross cognitive impairment at 5–6 months of age and could learn a simple visual discrimination task at a rate comparable to that of WT mice. This is in line with the lack of deficits previously reported in other cognitive assays at comparable ages. However, CVN mice did demonstrate a slower rate of reversal learning at these ages. Further exploration of this slowed reversal learning phenotype is planned using this highly translatable touch screen approach in order to identify deficits in other touch screen tasks at an early age, but also at later ages, when the AD-related phenotypes are more prominent in terms of brain pathology and behavioral readouts.