The central histaminergic neurons in vitro, and their neuroprotective role in excitotoxic cell death in the postnatal rat hippocampus.

Histamine acts as a neurotransmitter in the central nervous system. Brain histamine in synthesized in neurons located to the posterior hypothalamus, from where these neurons send their projections to different parts of the brain. Released histamine participates in the regulation of several physiological functions such as arousal, attention and body homeostasis. Disturbances in the histaminergic system have been detected in diseases such as epilepsy, sleep disorders, anxiety, depression, Alzheimer’s disease, and schizophrenia. The purpose of this thesis was to develop optimal culture conditions for the histaminergic neurons, to study their detailed morphology, and to find out their significance in the kainic acid (KA)-induced neuronal death in the immature rat hippocampus. The morphology of the histaminergic neurons in vitro was comparable with the earlier findings. Histamine-containing vesicles were found in the axon but also in the cell body and dendrites suggesting a possibility for the somatodendritic release. Moreover, histamine was shown to be colocalized with the vesicular monoamine transporter 2 (VMAT2) suggesting that VMAT2 transports histamine to the subcellular storage vesicles. Furthermore, histamine was localized with γ-aminobutyric acid (GABA) in distinct storage vesicles and with neuropeptide galanin partly in the same storage vesicles suggesting different corelease mechanisms for GABA and galanin with histamine. In the organotypic hippocampal slice cultures, KA-induced neuronal death was first detected 12 h after the treatment being restricted mainly to the CA3 subregion. Moreover, cell death was irreversible, since the 48 h recovery period did not save the cells, but instead increased the damage. Finally, neuronal death was suggested to be necrotic, since intracellular apoptotic pathways were not activated, and the morphological changes detected with the electron microscopy were characteristic for necrosis. In the coculture system of the hippocampal and posterior hypothalamic slices, histaminergic neurons significantly decreased epileptiform burst activity and neuronal death in the hippocampal slices, this effect being mediated by histamine 1 (H1) and 3 (H3) receptors. In conclusion, the histaminergic neurons were maintained succesfully in the in vitro conditions exhibiting comparable morphological characteristics as detected earlier in vivo. Moreover, they developed functional innervations within the hippocampal slices in the coculture system. Finally, the KA-induced regionspecific, irreversible and necrotic hippocampal pyramidal cell damage was significantly decreased by the histaminergic neurons through H1 and H3 receptors.

Hypoxia-associated biomarkers in rectal cancer treated by preoperative radiotherapy or chemoradiotherapy

Hypoksiaan liittyvät biologiset merkkiaineet leikkausta edeltävällä sädehoidolla tai kemosädehoidolla hoidetussa peräsuolisyövässä Peräsuolensyöpä on yleinen pahanlaatuinen kasvain. Leikkausta edeltävä sädehoito annetaan yleensä T3-T4-kasvaimille. Tutkimuksella pyrittiin selvittämään, voidaanko kasvaimen hapenpuutteeseen liittyvillä biologisilla merkkiaineilla arvioida peräsuolisyövän ennustetta leikkausta edeltävän sädehoidon tai kemosädehoidon jälkeen. Tällaisia merkkiaineita ovat hapenpuutteen vaikutuksesta aktivoituva HIF-1alfa hiilihappoanhydraasi IX (CA IX), sokerin kuljetukseen solussa osallistuva GLUT-1 sekä solun tukirankaproteiini ezrin. Tutkimukseen otettiin 178 potilasta, jotka olivat saaneet ennen leikkausta lyhyen (n=77) tai pitkän sädehoidon (n=10), pitkän sädehoidon ja solunsalpaajahoidon (n=37) tai ei mitään hoitoa (n=54). Lisäksi osalta leikkausta edeltävää sädehoitoa saaneelta potilaalta tutkittiin hoitoja edeltävät, diagnostiset näytteet (n=80). Tutkimuksessa käytettiin immunehistokemiallisia värjäysmenetelmiä. Kasvaimen regressiota (TRG) arvioitiin pitkän sädehoidon jälkeisistä näytteistä. Leikkausnäytteissä negatiivinen/heikko CA IX intensiteetti liittyi sekä pidempään tautispesifiseen (p=0.034) että tautivapaaseen elinaikaan (p=0.003) ja pitkän sädehoidon jälkeen HIF-1alfa-negatiivisuus pidempään tautispesifiseen (p=0.001) sekä negatiivinen/heikko GLUT-1 pidempään tautivapaaseen elinaikaan (p=0.066). Voimakas ezrin-ilmentymä diagnostisissa näytteissä liittyi lyhyempään tautivapaaseen ja tautispesifiseen (p=0.027 ja p=0.002) ennusteeseen. Monimuuttuja-analyysissä vahva CA IX intensiteetti leikkausnäytteissä ennusti itsenäisesti huonompaa tautivapaata ja tautispesifistä selviytymistä. Erinomainen TRG liittyi negatiiviseen/heikkoon CA IX- (p=0.057), ezrin- (p=0.012) ja GLUT-1 -ilmentymään (p=0.013) leikkausnäytteissä. Kun kaikki neljä merkkiainetta analysoitiin yhdessä monimuuttuja-analyysissä, CA IX intensiteetti leikkausnäytteissä ennusti itsenäisesti tautispesifistä elinaikaa. Voimakas CA IX-ilmentymä leikkausnäytteissä ja positiivinen HIF-1alfa- ja vahva GLUT-1-ilmentymä pitkän sädehoidon jälkeisissä leikkausnäytteissä sekä vahva ezrin-ilmentymä diagnostisissa näytteissä liittyivät epäsuotuisaan ennusteeseen. Monimuuttujaanalyysissä kohtalainen/voimakas CA IX intensiteetti leikkausnäytteissä ennusti itsenäisesti huonompaa tautivapaata ja tautispesifistä elinaikaa. CA IX on vahva biologinen merkkiaine peräsuolisyövässä.

On Glucose Metabolism in Patients with the m.3243A>G Mutation

Background: The m.3243A>G mutation in mitochondrial DNA is the most common cause for mitochondrial diabetes. In addition, unexpected deaths related to the m.3243A>G associate with encephalopathy and cardiomyopathy. Failing mitochondrial respiratory chain in neurons, myocytes and beta cells is considered to underlie the multiorgan manifestations of the m.3243A>G. Aims: The primary aim of the study was to characterize the organ-specific glucose metabolism in patients with m.3243A>G and secondly, to study patients with or without signs of diabetes, cardiomyopathy or encephalopathy. The insulin-stimulated glucose metabolism in brain, heart, skeletal muscle, adipose tissue and liver were measured with 2-deoxy-2-[18F]fluoro-α-D-glucose in 15 patients and 14 controls. Brain oxygen metabolism was assessed with [15O]oxygen and insulin secretion was modelled based on oral glucose tolerance test. Results: The glucose oxidation in brain was globally decreased in patients with or without clinical encephalopathy. The insulin-stimulated glucose influx to skeletal muscle and adipose tissue was decreased in patients with or without diabetes as the hepatic glucose metabolism was normal. Impaired beta cell function and myocardial glucose uptake were associated with the high m.3243A>G heteroplasmy. Conclusions: This cross-sectional study suggests that: 1) The ability of insulin to stimulate glucose metabolism in skeletal muscle and adipose tissue is weakened before the beta cell failure results in mitochondrial diabetes. 2) Glucose oxidation defect is detected in otherwise unaffected cerebral regions in patients with the m.3243A>G, thus it likely precedes the clinical encephalopathy. 3) Uneconomical glucose hypometabolism during hyperinsulinemia contributes to the cardiac vulnerability in patients with high m.3243A>G heteroplasmy

Starting Insulin Treatment in Type 2 Diabetes

Type 2 diabetes is a disorder of glucose metabolism characterized by chronic hyperglycemia. Initially type 2 diabetes is characterized by insulin resistance and impaired function of beta cells, leading progressively to insulin deficiency. Type 2 diabetes is treated with diet and other lifestyle changes, and with medication modulating e.g. insulin resistance, liver glucose production and insulin secretion. Injectable insulin is added to the treatment when lifestyle changes and other medication are insufficient to maintain adequate control of hyperglycemia. The aim of the treatment is to remove the symptoms of diabetes and to prevent late complications of diabetes. Insulin was traditionally started at hospital wards, but from the early 1990’s also in outpatient care. The first substudy of this thesis examined retrospectively initiation practices and how successfully insulin treatment was introduced in 1990 – 1996 in Southwestern Finland. This study aimed also at identifying the best methods of controlling plasma glucose. It showed that in the 1990’s the incidence of insulin treatment increased and was initiated more often in outpatient care than previously. The use of combination treatment also increased, first with sulfonylureas and later with metformin as the oral drug. In combination therapy the insulin dose was smaller than with insulin monotherapy. HbA1c improved similarly in middle-aged and older age groups. Weight increase associated with insulin initiation was smaller when combined with oral agents. A prospective insulin initiation study (1994 – 1998) tested the hypothesis that hyperglycemia (fasting and postprandial hyperglycemia) may affect the outcome of insulin initiation. The type of hyperglycemia was determined by the relation of fasting plasma glucose to HbA1c. Treatment was initiated with insulin Lente or human NPH insulin. In patients treated with insulin monotherapy twice daily the decline in HbA1c was markedly greater for postprandial than fasting hyperglycemia patients suggesting that hyperglycemia type has significance in the selection of the insulin regimen. Another insulin initiation study showed that patients with fasting hyperglycemia starting on insulin (2004-2005) were significantly more prone to overweight than patients with postprandial hyperglycemia. Irrespective of the insulin preparation (insulin NPH or insulin glargine), patients with fasting hyperglycemia had a greater weight increase compared to patients with postprandial hyperglycemia. Special attention should be paid to prevention of weight increase in these patients.