Synchrotron-generated microbeam sensorimotor cortex transections induce seizure control without disruption of neurological functions

Abstract

Synchrotron-generated X-ray microplanar beams (microbeams) are characterized by the ability to deliver extremely high doses of radiation to spatially restricted volumes of tissue. Minimal dose spreading outside the beam path provides an exceptional degree of protection from radio-induced damage to the neurons and glia adjacent to the microscopic slices of tissue irradiated. The preservation of cortical architecture following high-dose microbeam irradiation and the ability to induce non-invasively the equivalent of a surgical cut over the cortex is of great interest for the development of novel experimental models in neurobiology and new treatment avenues for a variety of brain disorders. Microbeams (size 100 µm/600 µm, center-to-center distance of 400 µm/1200 µm, peak entrance doses of 360-240 Gy/150-100 Gy) delivered to the sensorimotor cortex of six 2-month-old naïve rats generated histologically evident cortical transections, without modifying motor behavior and weight gain up to 7 months. Microbeam transections of the sensorimotor cortex dramatically reduced convulsive seizure duration in a further group of 12 rats receiving local infusion of kainic acid. No subsequent neurological deficit was associated with the treatment. These data provide a novel tool to study the functions of the cortex and pave the way for the development of new therapeutic strategies for epilepsy and other neurological diseases.

Figure 1. Isodose curves deposited by a microbeam.

Dose profiles deposited in a 16×16×16 cm³ water phantom by a single 100 µm wide and 10 mm high X-ray microbeam of 100 keV (Monte Carlo simulations), i.e. of energy similar to the median energy of the used spectrum (inset), propagating along the z direction The 50% of the dose is deposited at about 5 cm depth. Objects are not to scale.

Figure 2. Schematic representation of microbeam irradiation geometry.

Coronal (a) and sagittal (b) section of the Paxinos and Watson's rat brain atlas. Arrays of 100 or 600 µm thick microbeams (here represented the 100 µm case, minimum center-to-center spacing: 400 µm) were delivered perpendicular to the sensorimotor cortex using an atlas-based image guided X-ray setup. In (a) is indicated the point where the kainic acid injection was performed to create status epilepticus in rats. Copyright 1998 with permission from Elsevier.

Figure 3. Immunohistochemistry of phosphorylated Gamma-H2AX in the cortex of an irradiated rat.

Image shows the microbeam paths across the sensorimotor cortex of healthy rats: apoptotic neurons hit by microbeams (size: 100 µm, c-t-c 400 µm, incident dose 360 Gy) are evident.

Figure 4. Weight trend and rotarod performance of healthy non-irradiated and irradiated rats.

Data are means+S.E.M. (n = 3 for non-irradiated rats and n = 12 for irradiated rats).

Figure 5. Nissl staining of sensory motor cortex.

Nissl analysis performed 7 months after microbeams irradiation shows the lack of neurons along the microbeam path whereas neurons are spared in between microbeams (5b is a higher magnification of 5a).

Figure 6. Microbeams irradiations induce a reduction of seizure duration.

Duration of convulsive seizures was significantly reduced in rats undergoing transections either with 100 µm wide (n = 6) and 600 µm wide (n = 6) microbeams. A high dose (HD) and a low dose (LD) protocol were tested for both microbeam groups (n = 3 for each of the four irradiated groups and n = 6 for the non-irradiated control group). *p<0.05 (One-way ANOVA+Bonferroni's test) vs. non irradiated rats (Control). The differences among the irradiation groups were not statistically significant.

Figure 7. The control panel of the rat irradiation and imaging systems.

After an optical prepositioning of the target performed through 3 remote controlled video cameras (three upper panels), high resolution target determination is performed by X-ray imaging. A computer-guided robotic arm (Kappa goniometer, below the rat and not visible in the Figure) moves the stereotactic headframe to acquire radioscopic image necessary to individuate the bregma (radiographic image, bottom right) from which stereotactic coordinates allow the irradiation.