Institute of Fundamental Technological Research

The HIFU ablation technique is limited by the long duration of the procedure, which results from the large difference between the size of the HIFU beam’s focus and the tumor size. Ablation of large tumors requires treating them with a sequence of single HIFU beams, with a specific time interval in-between. The aim of this study was to evaluate the biological effects induced in a malignant solid tumor of the rat mammary gland, implanted in adult Wistar rats, during HIFU treatment according to a new ablation plan which allowed researchers to significantly shorten the duration of the procedure. We used a custom, automated, ultrasound imaging-guided HIFU ablation device. Tumors with a 1 mm thickness margin of healthy tissue were subjected to HIFU. Three days later, the animals were sacrificed, and the HIFU-treated tissues were harvested. The biological effects were studied, employing morphological, histological, immunohistochemical, and ultrastructural techniques. Massive cell death, hemorrhages, tissue loss, influx of immune cells, and induction of pro-inflammatory cytokines were observed in the HIFU-treated tumors. No damage to healthy tissues was observed in the area surrounding the safety margin. These results confirmed the efficacy of the proposed shortened duration of the HIFU ablation procedure and its potential for the treatment of solid tumors.

HIFU thermal ablation, breast cancer model, treatment plan, morphology, histology, ultrastructure, immune response, cell death, apoptosis, necrosis

An excessive glutamate level can result in excitotoxic damage and death of central nervous system (CNS) cells, and is involved in the pathogenesis of many CNS diseases. It may also be related to a failure of the blood-spinal cord barrier (BSCB). This study was aimed at examining the effects of extended administration of monosodium glutamate on the BSCB and spinal cord cells in adult male Wistar rats. The glutamate was delivered by subarachnoidal application of glutamate-carrying electrospun nanofiber mat dressing at the lumbar enlargement level. Half of the rats with the glutamate-loaded mat application were treated systemically with the histone deacetylase inhibitor valproic acid. A group of intact rats and a rat group with subarachnoidal application of an ‘empty’ (i.e., carrying no glutamate) nanofiber mat dressing served as controls. All the rats were euthanized three weeks later and lumbar fragments of their spinal cords were harvested for histological, immunohistochemical and ultrastructural studies. The samples from controls revealed normal parenchyma and BSCB morphology, whereas those from rats with the glutamate-loaded nanofiber mat dressing showed many intraparenchymal microhemorrhages of variable sizes. The capillaries in the vicinity of the glutamate-carrying dressing (in the meninges and white matter alike) were edematous and leaky, and their endothelial cells showed degenerative changes: extensive swelling, enhanced vacuo­lization and the presence of vascular intraluminal projections. However, endothelial tight junctions were generally well preserved. Some endothelial cells were dying by necrosis or apoptosis. The adjacent parenchyma showed astrogliosis with astrocytic hypertrophy and swelling of perivascular astrocytic feet. Neurons in the parenchyma revealed multiple symptoms of degeneration, including, inter alia, perikaryal, dendritic and axonal swelling, and destruction of organelles. All the damage symptoms were slightly less severe in the rats given valproic acid treatment, and were absent from both the intact rats and the rats with ‘empty’ nanofiber mat dressing. These results demonstrate that glutamate-loaded nanofiber mat dressing can locally create glutamate levels capable of damaging BSCB and that the resulting damage can be mitigated with concurrent systemic valproate treatment.

astrocyte, blood-spinal cord barrier, CNS damage, degeneration, endothelium, excitotoxicity, glutamate, neuron, valproate, vessels

Traumatic/surgical brain injury can initiate a cascade of pathological changes that result, in the long run, in severe damage of brain parenchyma and encephalopathy. Excessive scarring can also interfere with brain function and the glial scar formed may hamper the restoration of damaged brain neural pathways. In this preliminary study we aimed to investigate the effect of dressing with an L-lactide-caprolactone copolymer nanofiber net on brain wound healing and the fate of the formed glial scar. Our rat model of surgical brain injury (SBI) of the fronto-temporal region of the sensorimotor cortex imitates well the respective human neurosurgery situation. Brains derived from SBI rats with net-undressed wound showed massive neurodegeneration, entry of systemic inflammatory cells into the brain parenchyma and the astrogliosis due to massive glial scar formation. Dressing of the wound with the nanofiber net delayed and reduced the destructive phenomena. We observed also a reduction in the scar thickness. The observed modification of local inflammation and cicatrization suggest that nanofiber nets could be useful in human neurosurgery.

brain injury, L-lactide-caprolactone copolymer nanofiber net, glial scar, neurodegeneration

(Sub)chronic local drug application is clearly superior to systemic administration, but may be associated with substantial obstacles, particularly regarding the applications to highly sensitive central nervous system (CNS) structures that are shielded from the outer environment by the blood-brain barrier. Violation of the integrity of the barrier and CNS tissues by a permanently implanted probe or cannula meant for prolonged administration of drugs into specific CNS structures can be a severe confounding factor because of the resulting inflammatory reactions. In this study, we tested the utility of a novel way for (sub)chronic local delivery of highly active (i.e., used in very low amounts) drugs to the rat spinal cord employing a non-woven nanofiber mat dressing. To this end, we compared the morphology and motoneuron ( + ) counts in spinal cord cervical and lumbar segments between rats with glutamate-loaded nanofiber mats applied to the lumbar enlargement and rats with analogical implants carrying no glutamate. Half of the rats with glutamate-loaded implants were given daily valproate treatment to test its potential for counteracting the detrimental effects of glutamate excess. The mats were prepared in-house by electrospinning of an emulsion made of a solution of the biocompatible and biodegradable poly(L-lactide-co-caprolactone) polymer in a mixture of organic solvents, an aqueous phase with or without monosodium glutamate, and sodium dodecyl sulfate as an emulsifier; the final glutamate content was 1.4 µg/mg of the mat. Three weeks after mat implantation there was no inflammation or considerable damage of the spinal cord motoneuron population in the rats with the subarachnoid dressing of a glutamate-free mat, whereas the spinal cords of the rats with glutamate-loaded nanofiber mats showed clear symptoms of excitotoxic damage and a substantial increase in dying/damaged motoneuron numbers in both segments studied. The rats given systemic valproate treatment showed significantly lower percentages of damaged/dying motoneurons in their lumbar enlargements. These results demonstrate the capacity of nanofiber mats for generation of neurotoxic glutamate in the rat CNS. However, the tested nanofiber mats need further improvements aimed at extending the period of effective drug release and rendering the release more steady.

CNS injury, electrospinning, excitotoxicity, glutamate, motoneuron, nanofibers, neurodegeneration, spinal cord, valproate

Excessive cicatrisation or epidural fibrosis in the operative field is an inappropriate event occasionally occurring after neurosurgical procedures (i.e., spine procedures and craniotomies). This excessive process may disturb the postopera­tive course and render reoperations more difficult and risky. The literature describes this phenomenon as accompanying up to 20% of neurosurgical procedures. The scar tissue that forms postoperatively adheres to the dura mater, penetrates into the spinal canal and can cause narrowing symptoms, neurological deficits and pain. The incidence and spread of this excessive scar or epidural fibrosis can be prevented through the modification of the surgical technique by incorporating endoscopic or microscopic access to minimize the operative field and the use of isolating substances (autogenous or heterogeneous) administered intraoperatively.

The aim of this experimental study was to morphologically assess the cicatrisation process, adhesion and to prevent excessive scar formation with the local use of membranes manufactured by an electrospinning process (nanotechnology). We also investigated whether the biodegradable nanofibrous net triggers or modifies the immunological response or the local inflammatory process.

Micro-nanofibrous membranes were produced by the electrospinning process. A biodegradable, medically certified copolymer poly(L-lactide-co-caprolactone) (PLCL) was used as the electrospun material.

An experimental rat model was used in this study. Experimental and control groups were formed with specified follow-up times of 4, 14 and 30 days. During the operation, a two-level laminectomy in the thoracic segment was performed. The operative field was divided into two regions. Isolating material was used on the dura mater and surface of the spinal cord in the area where the laminectomy was performed. The material was analysed with the use of light and electron microscopy.

Local cicatrisation can be modified using nanomaterials. Scar formation and epidural fibrosis can be limited and modi­fied locally. No local inflammation process was observed.

Initial observations indicate the potential for the effective use of materials obtained in the electrospinning process to prevent cicatrisation.

neurosurgery, cicatrisation, epidural fibrosis, copolymer, poly(L-lactide-co-caprolactone), nanofibrous net, rat model

Introduction:
Introduction of nanotechnology into medicine has provided new therapeutic options. It has been demonstrated that implantation of nanofiber mats after nervous system injury allowed to diminish scar size and infl ammatory reaction. It is also possible that, due to their ability to release active factors, nanofiber mats may replace intracerebral probes. To assess potential usefulness of nanofiber mats in releasing active substances we implanted them into the spinal cord subarachnoid space in adult Wistar rats.

Material and Method:
The experimental animals were divided into four groups: group 1 – rats with implanted nanofiber mats, group 2 – rats with implanted nanofiber mats releasing glutamate, group 3 – rats with nanofiber mats releasing glutamate and treated orally by sodium valproate, and group 4 – control animals without nanofiber mats. The animals were killed 21 days after the mats implantation. Then, histopathological, immunohistochemical and ultrastructural evaluation of the spinal cords was performed.

Results:
Morphological assessment revealed that implantation of nanofi ber mats caused neither spinal cord damage nor inflammation (group 1). Also nanofi ber mats releasing glutamate did not produce inflammatory reaction (group 2 and 3) although in group 2 morphological changes indicating toxic influence of glutamate were observed. These changes were less severe in group 3.

Conclusions:
(1) Nanofi ber mats are biocompatible and can be useful in long-term animal experiments. (2) Nanofi ber mats are able to release glutamate into the subarachnoid space. (3) Sodium valproate has a protective influence against glutamate toxicity.

electrospinning, nanofibers, drug delivery, neurology, sodium glutamate, animal model