Joanna Lewin-Kowalik

Total ghrelin plasma level in patients with the restrictive type of anorexia nervosa.

Ghrelin is produced mainly in the stomach and is an essential link of the brain-gut axis. Ghrelin stimulates hunger centers in hypothalamus controlling food intake and body mass gain. The aim of the study is to analyze the total ghrelin plasma level in patients suffering from restrictive type of anorexia nervosa (AN-R). According to DSM-IV classification a group of 30 AN-R patients was investigated before and after 3 and 6 months of therapy. Therapy included normocaloric diet and cognitive-behavioral psychotherapy (CBT). The control group consisted of 20 girls without any eating disorders. Before the therapy the total ghrelin plasma level in AN-R patients was significantly higher than in the control group. After 3 and 6 months of treatment the total ghrelin plasma level in AN-R patients was significantly lower than in the control group. In AN-R patients, the total ghrelin plasma level is connected with the pathological feeding behavior.

Cardiac Rhabdomyomas in Tuberous Sclerosis Complex Show Apoptosis Regulation and mTOR Pathway Abnormalities

Cardiac rhabdomyoma (CR) is the most common heart tumor in children and is usually associated with tuberous sclerosis complex (TSC). Tuberous sclerosis complex is a genetic disorder caused by a mutation in either of 2 genes (TSC1 or TSC2) and characterized by the formation of hamartomas in multiple organs. The 2 TSC proteins, hamartin and tuberin, antagonize the mammalian target of rapamycin (mTOR) signaling pathway, thus regulating cell growth and proliferation. Recently, some trials treating TSC with the mTOR inhibitor rapamycin have been published; however, the impact of such treatment on heart tumors is not known. The aim of the present paper was to study the molecular pathobiology of CRs. Six CR samples were studied. The expression of S6K1, pErk, Erk, Akt, pAkt, 4E-BP1, hamartin, tuberin, mTOR, bcl-2, Bax, and Ki-67 was examined using immunohistochemistry and Western blot methods. Increased expression of Bax, mTOR, pS6K, pErk, and 4E-BP1 was found in all CR samples. Hamartin and tuberin expression was decreased in tumors versus normal heart tissues. This is the first study showing mTOR pathway dysregulation and an increased expression of proapoptotic Bax protein in CRs associated with TSC.

Regeneration of sciatic nerves of adult rats induced by extracts from distal stumps of pre-degenerated peripheral nerves

Despite numerous experimental and clinical attempts to reconstruct injuries of peripheral nerves, the methods developed until now have not been sufficiently effective. We examined the influence of extracts (postmicrosomal fractions) obtained from non‐pre‐degenerated or 7‐day‐pre‐degenerated distal segments of peripheral nerves on the regeneration of injured sciatic nerves of male adult rats. The extracts were introduced to the site of injury with autologous connective tissue chambers filled with fibrin. Reference groups were treated with brain‐derived neurotrophic factor (BDNF) or fibrin only. We examined DiI‐labeled motoneurons, toluidine blue‐labeled myelinated fibers in the mid‐part of the chambers, and AChE‐positive nerve endings to assess the regeneration intensity. In addition, the length of fibers regrowing within the chambers was measured. We found that extracts obtained from distal stumps of 7‐day‐pre‐degenerated peripheral nerves enhanced nerve regeneration as strongly as BDNF.

Tuberin and hamartin expression is reduced in the majority of subependymal giant cell astrocytomas in tuberous sclerosis complex consistent with a two-hit model of pathogenesis

Subependymal giant cell astrocytomas are distinctive brain tumors that are seen only in tuberous sclerosis complex. Although histologically benign, they cause both moribidity and occasional mortality owing to progressive growth in some patients. Tuberous sclerosis complex is an autosomal dominant genetic disorder with a high sporadic case rate that is due to mutations in either of two genes, TSC1 and TSC2, encoding hamartin and tuberin, respectively. The pathogenesis of subependymal giant cell astrocytomas in tuberous sclerosis complex is uncertain. In this study, we examined the expression of tuberin and hamartin in subependymal giant cell astrocytomas from nine patients with tuberous sclerosis complex by immunohistochemistry with confocal microscopy. Loss of hamartin expression was seen in all subependymal giant cell astrocytomas, including five from patients with germline TSC2 mutations and two from patients with germline TSC1 mutations. The subependymal giant cell astrocytomas of six of nine patients had no expression of tuberin as well, whereas three patients retained some tuberin expression. Tuberin expression was seen in one patient with a TSC2 germline mutation and two patients whose mutational status was not determined. Overall, these data indicate a loss of both tuberin and hamartin expression in the subependymal giant cell astrocytomas of patients with both TSC1 and TSC2 mutations and are consistent with a two-hit disease pathogenesis model for the development of subependymal giant cell astrocytomas. (J Child Neurol 2004;19:102—106).

Overexpression of copper/zinc-superoxide dismutase in transgenic mice markedly impairs regeneration and increases development of neuropathic pain after sciatic nerve injury

Despite the general capacity of peripheral nervous system to regenerate, peripheral nerve injury is often followed by incomplete recovery of function, sometimes with the burden of neuropathic pain. The mechanisms of both regeneration and nociception have not been clarified, but it is known that inflammatory reactions are involved. Cu/Zn‐superoxide dismutase (SOD1) is an important scavenger protein that acts against oxidative stress. It has been shown to play an important role in apoptosis and inflammation. The aim of this study was to examine the role of SOD1 overexpression in peripheral nerve regeneration and neuropathic pain‐related behavior in mice. Sciatic nerves of SOD1‐overexpressing and FVB/N wild type‐mice were transected and immediately resutured. Evaluation of motor and sensory function and autotomy was carried out during 4 weeks of followup. We found markedly worse sciatic function index outcome as well as more significant atrophy of denervated muscles in SOD1‐overexpressing animals compared with wild type. Autotomy was markedly worse in SOD1 transgenic mice than in wild‐type animals. Histological evaluation revealed that the intensity of regeneration features, including numbers of GAP‐43‐positive growth cones, Schwann cells, and macrophages in the distal stump of the transected nerve, was also decreased in transgenic mice. Neuroma formation at the injury site was significantly more prominent in this group. Taken together, our findings suggest that SOD1 overexpression is deleterious for nerve regeneration processes and aggravates neuropathic pain‐like state in mice. This can be at least partially ascribed to disturbed inflammatory reactions at the injury site.

Interleukin-1β Increases Release of Endothelin-1 and Tumor Necrosis Factor as Well as Reactive Oxygen Species by Peripheral Leukocytes During Experimental Subarachnoid Hemorrhage

In the subarachnoid hemorrhage (SAH) blood mixes with cerebrospinal fluid, what starts immunoinflammatory processes - increased synthesis of proinflammatory cytokines, and formation of reactive oxygen species (ROS), resulting in pre-activation of different populations of peripheral leukocytes. Migration of leukocytes to the brain parenchyma through broken blood brain barrier may produce extra brain tissue injury besides of that resulting from SAH. We examined in adult rats the effect of interleukin-1β (IL-1β) neutralization on secretion of cytokines as well as production of ROS in the course of SAH. SAH was produced by injection of 150 μL of autologous arterial blood into cisterna magna. In 50% of animals, IL-1beta activity was inhibited by intracerebroventricular administration of anti-rat IL-1β antibodies. Ninety minutes or 24 hrs following surgery, blood samples were drawn from the extraorbital plexus and centrifuged to obtain two leukocyte subpopulations - polymorphonuclear (PMN) and mononuclear (MN). The chemiluminescence, a hallmark of ROS synthesis, was measured in PMNs. In supernatants from MNs cultures, concentrations endothelin-1 (ET-1), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were assessed. SAH caused the increase ofn PMNs chemiluminescence as well as the increase of production of ET-1 and TNF-α by MNs but had no influence on IL-6 concentration. Neutralization of IL-1β resulted in significant decrease of chemiluminescence as well as concentration of both ET-1 and TNF-α, while IL-6 concentration was increased. These revealed an important role of IL-1β in the activation of peripheral leukocytes in the course of subarachnoid hemorrhage.

A rat experimental model of glaucoma incorporating rapid-onset elevation of intraocular pressure.

Glaucoma is a chronic disease that causes structural and functional damage to retinal ganglion cells (RGC). The currently employed therapeutic options are not sufficient to prevent vision loss in patients with glaucoma; therefore, there is a need to develop novel therapies, which requires the creation of functional, repeatable and easy-to-utilize animal models for use in pre-clinical studies. The currently available models ensure only low to moderate damage in optic nerves, with high variation in the outcomes and poor repeatability. We have developed an effective and reproducible rat glaucoma model based on a previous idea for a “Bead Model” in mice, which could be useful in future glaucoma research. Additionally, in an attempt to achieve rapid elevation of Intraocular Pressure (IOP), we included an initial “high-pressure injury” as part of this method, which serves as the equivalent of a severe glaucoma attack. These modifications made it possible to achieve longer lasting IOP elevation with chronic damage of retinal ganglion cells.

Reduction of post-traumatic neuroma and epineural scar formation in rat sciatic nerve by application of microcrystallic chitosan

Injury of peripheral nerve is associated with the development of post‐traumatic neuroma at the end of the proximal stump, often being the origin of neuropathic pain. This type of pain is therapy‐resistant and therefore extremely nagging for patients. We examined the influence of the microcrystallic chitosan gel applied to the proximal stump of totally transected sciatic nerve on the neuroma formation and neuropathic pain development in rats. In 14 rats, right sciatic nerve was transected and the distal stump was removed to avoid spontaneous rejoining. In the chitosan (experimental) group (n = 7), the proximal stump was covered with a thin layer of the microcrystallic chitosan gel. In control animals (n = 7), the cut nerve was left unsecured. Autotomy, an animal model of neuropathic pain, was monitored daily for 20 weeks following surgery. Then, the animals were perfused transcardially and the proximal stumps of sciatic nerves were dissected and subjected to histologic evaluation. The presence, size, and characteristics of neuromas as well as extraneural fibrosis were examined. In chitosan group, the incidence and the size of the neuroma were markedly reduced, as compared with the control group; however, there was no difference in autotomy behavior between groups. In addition, extraneural fibrosis was significantly reduced in chitosan group when compared to the control group. The results demonstrate beneficial influence of microcrystallic chitosan applied to the site of nerve transection on the development of post‐traumatic neuroma and reduction of extraneural fibrosis, however without reduction of neuropathic pain.

Predegenerated Peripheral Nerve Grafts Rescue Retinal Ganglion Cells from Axotomy-Induced Death

The inability of axons to grow across damaged central nervous system tissue is a well-known consequence of injury to the brain and spinal cord of adult mammals. Our previous studies showed that predegenerated peripheral nerve grafts facilitate neurite outgrowth from the injured hippocampus and that this effect was particularly distinct when 7-, 28-, and 35-day-predegenerated nerve grafts were used. The purpose of the present study was to use the above method to induce and support the regrowth of injured nerve fibers as well as the survival of retinal ganglion cells (RGCs). Adult Sprague-Dawley rats were assigned to three groups. In the experimental groups transected optic nerve was grafted with peripheral nerve (predegenerated for 7 days (PD) or nonpredegenerated). In the control group, the optic nerve was totally transected. RGCs and growing fibers labeled with fluorescent tracers were examined. They were counted and the results were subjected to statistical analysis. Retinal ganglion cells survived in the groups treated with predegenerated as well as nonpredegenerated grafts; however, the number of surviving retinal ganglion cells was significantly higher in the first one. In both groups the regrowth of the transected optic nerve was observed but the distance covered by regenerating fibers was longer in the PD group. No fibers inside grafts and no labeled cells in retinas were present in the control animals. On the basis of the obtained results we can state that the predegeneration of grafts enhance their neurotrophic influence upon the injured retinal ganglion cells.

Time-dependent regenerative influence of predegenerated nerve grafts on hippocampus

Our previous studies have revealed that the predegeneration facilitated the neurite outgrowth from hippocampus following the peripheral nerve grafts implantation. The aim of the present work is to find whether the stimulative power of peripheral nerve grafts depends on the time lapse after the transection. Autologous predegenerated distal stumps of the rat sciatic nerves were implanted into the hippocampus on the 7th, 14th, 28th, and 35th day following the transection. Six weeks later, horseradish peroxidase conjugated with fluorescein isothiocyanate was injected into the graft and frozen sections of brains were made. Fluorescence microscope examination has shown that FITC-HRP labeled cells were present among the hippocampal neurons in all the brains under examination, excluding these grafted with 14-day predegenerated peripheral nerves. The FITC-HRP labeled neurons were particularly numerous when the 7- and 35-day-old predegenerated stumps were used as grafts.