Alternative Anesthesia of Neonatal Mice for Global rAAV Delivery in the Brain With Non-detectable Behavioral Interference in Adults.

Viral-transduced gene expression is the current standard for cell-type-specific labeling and cell tacking in experimental neuroscience. To achieve widespread gene expression, a viral delivery method to neonatal rodents was introduced more than two decades ago. Most of those neonatal viral vector injection-based gene transduction methods in mice used deep hypothermia for anesthesia, which was reported to be associated with behavioral impairments. To explore other options for neonatal viral applications, we applied a combination of Medetomidine, Midazolam, and Fentanyl (MMF), each of which can be antagonized by a specific antagonist. Later in their adulthood, we found that adult mice, that received the MMF-induced anesthesia, combined with virus-injected into the brain at postnatal day 2, showed similar performance in all behavioral tasks tested, including tasks for motor coordination, anxiety-related tasks, and spatial memory when compared to adult naïve littermates. This demonstrates that MMF anesthesia could be safely applied to mice for neonatal viral transduction at P2.

The effects of neonatal cryoanaesthesia-induced hypothermia on adult emotional behaviour and stress markers in C57BL/6 mice.

Since the early 1930s, deep hypothermia (cryoanaesthesia) has been a useful anaesthetic in several types of surgery on neonatal rodents. Especially against the background of modern techniques in systems neuroscience, the method enjoys again increasing popularity. However, little is known about its effects on the subsequent adult behavioural and physiological profile. To systematically investigate the effects of neonatal cryoanaesthesia on adult basal and emotional behaviour as well as on physiological development, 59 C57BL/6 mouse pups were randomly assigned to one of three treatment groups: Pups of the first group were exposed to the hypothermia treatment (H) on postnatal day 3, while pups of the other two groups served as controls: These pups either remained in the home cage without any intervention (C), or were separated from the mother for 15 min (MS) to differentiate between effects of neonatal isolation alone versus hypothermia that inevitably goes along with neonatal isolation. Subsequent behavioural analyses were conducted during adulthood (P 84-P 130), including tests for exploratory, anxiety-like and depression-like behaviour. At the age of about 145 days mice were decapitated to record BDNF levels in the hippocampus and serum corticosterone. Altogether, H mice were found to display slightly increased anxiety levels on the O-Maze, but did not differ from the control animals in any other behavioural test. Subtle alterations in anxiety-like behaviour, however, were not accompanied by physiological changes in serum corticosterone and hippocampal BDNF levels, arguing against an overall long-lasting effect of neonatal hypothermia on the emotional profile of adult mice.

Device for the local radiation of tumor-bearing laboratory rodents.

Since no standard method for reproducibly irradiating local tumors of laboratory rodents exists, the authors designed a device to accurately irradiate rodent tumors. Laboratory personnel can easily assemble this inexpensive device. The new device delivers highly focused treatment and avoids the need for anesthesia and sedation. It represents an improvement over former improvised procedures done at the authors' institutions, because it allows for more accurate irradiation of rodent tumors. The authors used the device to irradiate single tumors on the left hind legs of 60 mice and 10 rats with a dosage of 7 gray (Gy) ionizing radiation. At time of irradiation, the clinical condition of the animals was very poor, but all animals survived the treatment.

Efficacy and tolerability of an aminopterin-albumin conjugate in tumor-bearing rats.

The antifolate aminopterin (AMPT) was developed before methotrexate (MTX), but was not clinically established or generally used due its increased toxicity compared to MTX. Recently, we reported on the increased metabolism of albumin conjugates such as methotrexate-albumin (MTX-SA) in malignant tumors and the feasibility of using albumin as a carrier for drug targeting. Consequently, AMPT was covalently bound to serum albumin (AMPT-SA) at a 1:1 molar ratio. Biodistribution, tolerability and efficacy of this novel conjugate were studied in Walker-256 (W-256) carcinoma-bearing rats. As compared to native albumin, the same biodistribution and plasma clearance were found for AMPT-SA, which achieved 20.1% tumor uptake (estimated uptake per g tumor 6.4%) within 24 h after i.v. administration in rats. In a randomized study, AMPT-SA, repeatedly i.v. injected, was compared with low-molecular-weight AMPT. Depending on the molar concentration, the maximum tolerated dose (MTD) of AMPT covalently bound to SA was twice that of unbound AMPT (three repeated injections of 1.0 mg AMPT-SA/kg body weight versus three repeated injections of 0.5 mg AMPT/kg body weight; p=0.0006). Efficacy was studied at the level of the MTD and MTD/2, and demonstrated that AMPT-SA was significantly more active. At the MTD/2 in W-256 carcinoma-bearing rats, AMPT-SA achieved a 100% volume reduction and an optimal volume reduction during treatment/control (T/C) of 8.3% compared to a 53% volume reduction of AMPT and a T/C of 16.5% (p=0.032). Tumor relapses were reduced and occurred later in the AMPT-SA group (two tumor recurrences for AMPT-SA versus seven for AMPT; p=0.05). In this comparative study, the AMPT-SA conjugate showed high antitumor activity in vivo and a favorable toxicity compared to low-molecular-weight AMPT. These effects are attributed to the albumin carrier which seems to be an effective tool for selective tumor drug targeting.

The RAG 2 mouse model for xenografted human oral squamous cell carcinoma.

Animal tumor models are still essential for the development of new medication and therapy concepts. In the field of human oral squamous cell cancer, there are few reliable xenografted tumor models available. Therefore, during a two-course experiment, we established a new xenografted tumor model of human oral squamous cell cancer. The tumor growth rates of two different tumor cell lines were compared in the inbred immunodeficient CD-17-RAG 2 mouse, NMRI-SCID mouse (scid/scid), and Swiss nude mouse (nu/nu). The tumor cell line from a lymphnode metastasis of an oral squamous cell carcinoma (XF 354) had a faster growth rate and a more characteristic histology than did the cell line from the primary tumor of a squamous cell carcinoma of the floor of the mouth (UM-SCC-14C). The highest tumor growth rate was observed in the RAG 2 mouse, followed by the SCID mouse. The Swiss nude mouse showed no tumor growth. The combination of the XF 354 tumor cell line and the RAG 2 mouse was most successful, with a tumor growth rate of 95%. Our animal model is very reliable and allows manipulations for as long as 30 min under anesthesia outside of microbiologic safety cabinets, where the handling of animals is much more comfortable and less time-consuming. The tumor histology was easily interpreted by using light microscopy. Steps for cell cultivation and tumor implantation are described and discussed. Therefore we strongly recommend the use of the model comprising the RAG 2 mouse with the xenografted cell line XF 354 for research in the field of human oral squamous cell carcinoma.