Neutrophil Depletion Enhanced the Clostridium novyi-NT Therapy in Mouse and Rabbit Tumor Models

Background Hypoxia is a prominent feature of solid tumors and can function as fertile environment for oncolytic anaerobic bacteria such as Clostridium novyi-NT (C. novyi-NT) where it can induce tumor destruction in mice and patients. However, two major obstacles have limited its use, namely the host inflammatory response and the incomplete clearance of normoxic tumor areas. Methods In this study, we first used a subcutaneous tumor model of a glioblastoma (GBM) cell line in immunocompetent mice to investigate the local distribution of tumor hypoxia, kinetics of C.novyi-NT germination and spread, and the local host immune response. We subsequently applied the acquired knowledge to develop a C.novyi-NT therapy in an orthotopic rabbit brain tumor model. Results We found that local accumulation of granular leukocytes, mainly neutrophils, could impede the spread of bacteria through the tumor and prevented complete oncolysis. Depletion of neutrophils via anti-Ly6G antibody or bone marrow suppression using hydroxyurea significantly improved tumor clearance. We then applied this approach to rabbits implanted with an aggressive intracranial brain tumor and achieved long term survival in majority of the animals without apparent toxicity. Conclusion These results indicated that depleting neutrophils can greatly enhance the safety and efficacy of C.novyi-NT cancer therapy for brain tumors.

Spectral Optical Properties of Rabbit Brain Cortex between 200 and 1000 nm

The knowledge of the optical properties of biological tissues in a wide spectral range is highly important for the development of noninvasive diagnostic or treatment procedures. The absorption coefficient is one of those properties, from which various information about tissue components can be retrieved. Using transmittance and reflectance spectral measurements acquired from ex vivo rabbit brain cortex samples allowed to calculate its optical properties in the ultraviolet to the near infrared spectral range. Melanin and lipofuscin, the two pigments that are related to the aging of tissues and cells were identified in the cortex absorption. By subtracting the absorption of these pigments from the absorption of the brain cortex, it was possible to evaluate the true ratios for the DNA/RNA and hemoglobin bands in the cortex—12.33-fold (at 260 nm), 12.02-fold (at 411 nm) and 4.47-fold (at 555 nm). Since melanin and lipofuscin accumulation increases with the aging of the brain tissues and are related to the degeneration of neurons and their death, further studies should be performed to evaluate the evolution of pigment accumulation in the brain, so that new optical methods can be developed to aid in the diagnosis and monitoring of brain diseases.

Spectral Optical Properties of Rabbit Brain Cortex Between 200 and 1000 nm

The knowledge of the optical properties of biological tissues in a wide spectral range is highly important for the development of noninvasive diagnostic or treatment procedures. The absorption coefficient is one of those properties, from which various information about tissue components can be retrieved. Using transmittance and reflectance spectral measurements acquired from ex vivo rabbit brain cortex samples, allowed to calculate its optical properties in the ultraviolet to the near infrared spectral range. Melanin and lipofuscin, the two pigments that are related to the ageing of tissues and cells were identified in the cortex absorption. By subtracting the absorption of these pigments from the absorption of the brain cortex, it was possible to evaluate the true ratios for the DNA/RNA and hemoglobin bands in the cortex – 12.33 fold (at 260 nm), 12.02 fold (at 411 nm) and 4.47 fold (at 555 nm). Due to the fact that the accumulation of melanin and lipofuscin increases with the ageing of the brain tissues and are related to the degeneration of neurons and their death, further studies should be performed to evaluate the evolution of pigment accumulation in the brain to prevent the development of Alzheimer, Parkinson and stroke pathologies in the brain.

Development of a photochemical thrombosis investigation system to obtain a rabbit ischemic stroke model

Photochemical thrombosis is a method for the induction of ischemic stroke in the cerebral cortex. It can generate localized ischemic infarcts in the desired region; therefore, it has been actively employed in establishing an ischemic stroke animal model and in vivo assays of diagnostic and therapeutic techniques for stroke. To establish a rabbit ischemic stroke model and overcome the shortcoming of previous studies that were difficult to build a standardized photothrombotic rabbit model, we developed a photochemical thrombosis induction system that can produce consistent brain damage on a specific area. To verify the generation of photothrombotic brain damage using the system, longitudinal magnetic resonance imaging, 2,3,5-triphenyltetrazolium chloride staining, and histological staining were applied. These analytical methods have a high correlation for ischemic infarction and are appropriate for analyzing photothrombotic brain damage in the rabbit brain. The results indicated that the photothrombosis induction system has a main advantage of being accurately controlled a targeted region of photothrombosis and can produce cerebral hemisphere lesions on the target region of the rabbit brain. In conjugation with brain atlas, it can induce photochemical ischemic stroke locally in the part of the brain that is responsible for a particular brain function and the system can be used to develop animal models with degraded specific functions. Also, the photochemical thrombosis induction system and a standardized rabbit ischemic stroke model that uses this system have the potential to be used for verifications of biomedical techniques for ischemic stroke at a preclinical stage in parallel with further performance improvements.

Glutamic Acid Decarboxylase Concentration Changes in Response to Stress and Altered Availability of Glutamic Acid in Rabbit (Oryctolagus cuniculus) Brain Limbic Structures

Glutamic acid decarboxylase (GAD) is an enzyme that catalyses the formation of γ-aminobutyric acid (GABA), the most important inhibitory neurotransmitter, from glutamic acid (Glu), which is considered the most important excitatory transmitter in the central and peripheral nervous systems. GAD is a key enzyme that provides a balance between Glu and GABA concentration. Hence, it can be assumed that if the GAD executes the synthesis of GABA from Glu, it is important in the stress response, and thus also in triggering the emotional states of the body that accompany stress. The aim of the study was to investigate the concentration of the GAD in motivational structures in the brain of the rabbit (Oryctolagus cuniculus) under altered homeostatic conditions caused by stress and variable availability of Glu. Summarising, the experimental results clearly showed variable concentrations of GAD in the motivational structures of the rabbit brain. The highest concentration of GAD was found in the hypothalamus, which suggests a strong effect of Glu and GABA on the activity of this brain structure. The GAD concentrations in individual experimental groups depended to a greater extent on blocking the activity of glutamate receptors than on the effects of a single stress exposure. The results obtained clearly support the possibility that a rapid change in the concentration of GAD could shift bodily responses to quickly achieve homeostasis, especially in this species. Further studies are necessary to reveal the role of the Glu–GAD–GABA system in the modulation of stress situations as well as in body homeostasis.

Research on the measurement of intracranial hemorrhage in rabbits by a parallel-plate capacitor

Intracranial hemorrhage (ICH) carrying extremely high morbidity and mortality can only be detected by CT, MRI and other large equipment, which do not meet the requirements for bedside continuous monitoring and pre-hospital first aid. Since the biological tissues have different dielectric properties except the pure resistances, and the permittivity of blood is far larger than that of other brain tissues, here a new method was used to detect events of change at the blood/tissue volume ratio by measuring of the head permittivity. In this paper, we use a self-made parallel plate capacitor to detect the intracranial hemorrhage in rabbits by contactless capacitance measurement. The sensitivity of the parallel-plate capacitor was also evaluated by the physical solution measurement. The results of physical experiments show that the capacitor can distinguish between three solutions with different permittivity, and the capacitance increased with the increase of one solution between two plates. At the next step in the animal experiment, the capacitance changes caused by 2 ml blood injection into the rabbit brain were measured. The results of animal experiments show that the capacitance was almost unchanged before and after the blood injection, but increased with the increase of the blood injection volume. The increase of capacitance caused by blood injection was much larger than that before and after blood injection (P < 0.01). The experiments show that this method is feasible for the detection of intracranial hemorrhage in a non-invasive and contactless manner.