Caroline Gahm

Temporal profiles and cellular sources of three nitric oxide synthase isoforms in the brain after experimental contusion.

OBJECTIVE Nitric oxide (NO) is a universal mediator of biological effects in the brain. It has been implicated in the pathophysiological processes of traumatic brain injury. Understanding its pathophysiological role in vivo requires an understanding of the cellular sources and tissue compartments of the differentially regulated NO synthase (NOS) isoforms. This study was undertaken to investigate the cellular sources and tissue compartments of NO produced after experimental brain contusions in rats, by analysis of the early expression of the three isoforms of NOS, i.e., the inducible, endothelial, and neuronal isoforms. METHODS Focal brain contusions were produced in 24 rats using a weight-drop model. The animals were killed 6, 12, 24, 36, or 48 hours after trauma. Sections were analyzed by immunohistochemical and immunofluorescence analyses. Double staining assays were used to define which cells produced the different NOS isoforms. RESULTS Increases in endothelial NOS-, inducible NOS (iNOS)-, and neuronal NOS-positive cells were detectable by 6 hours after trauma. Endothelial NOS and iNOS levels peaked at 6 and 12 hours, respectively. Expression of neuronal NOS initially increased to a peak at 12 hours but then decreased to a level lower than that in control samples at 36 hours. Endothelial NOS was expressed exclusively in endothelial cells, whereas iNOS was expressed in neutrophils and macrophages. Neuronal NOS was predominantly detected in neurons but was also unexpectedly detected in polymorphonuclear cells. CONCLUSION In this model, the most striking finding regarding NO-producing enzymes was the expression of iNOS in polymorphonuclear cells and macrophages, cells that invade injured brain tissue. iNOS is thus implicated as a therapeutic target in contusional injuries. This pattern of NOS expression cannot be generalized to all types of brain injuries. The different compartments and cells that can produce NO are differentially regulated; therefore, compartmentalization can explain why NO is beneficial or detrimental, depending on the circumstances. A knowledge of different potential sites and sources of NO is required for any attempts to interfere with the pathophysiological properties of NO.

Sustained Inflammation Due to Nuclear Factor-Kappa B Activation in Irradiated Human Arteries

OBJECTIVES The aim of this study was to investigate gene expression networks related to cardiovascular disease in radiated human arteries. BACKGROUND Recent epidemiological studies have shown that radiotherapy is associated with cardiovascular disease years after treatment. However, the molecular mechanisms underlying late effects of radiation are poorly described. METHODS Arterial biopsies from radiated and nonradiated human conduit arteries, from the same patient, were simultaneously harvested during microvascular free tissue transfer for cancer-reconstruction in 13 patients, 4 to 500 weeks from radiation treatment. Radiated and nonradiated arteries were compared, with Affymetrix (Santa Clara, California) microarrays on a subset of the material to generate candidate genes. A Taqman (Applied Biosystems, Foster City, California) low-density array of 45 selected genes was designed for analysis of the whole material. RESULTS Thirteen genes were synchronously expressed in all patients (p = 0.0015), including CCL8, CCL3, CXCL2, DUSP5, FGFR2, HMOX1, HOXA9, IL-6, MMP-1, PTX3, RDH10, SOD2, and TNFAIP3. A majority of differentially regulated genes related to the nuclear factor-kappa B (NF-kappaB) signaling pathway and were dysregulated even years after radiation. The NF-kappaB activation was confirmed by immunohistochemistry and immunofluorescence. CONCLUSIONS In the present study, we found sustained inflammation due to NF-kappaB activation in human radiated arteries. The results are supported by previous in vitro findings suggesting that deoxyribonucleic acid injury, after radiation, activates NF-kappaB. We also suggest that HOXA9 might be involved in the regulation of NF-kappaB activation. The observed sustained inflammatory response can explain cardiovascular disease years after radiation.

Nitric oxide synthase expression after human brain contusion.

OBJECTIVE Nitric oxide is a universal mediator of biological effects in the brain, and it has been implicated in the pathophysiological processes of traumatic brain injury. Experimental studies have indicated posttraumatic up-regulation of the three different isoforms of nitric oxide synthase (NOS) (i.e., inducible NOS [iNOS], endothelial NOS, and neuronal NOS) after brain trauma. This study was undertaken to investigate the cellular sources and tissue compartments of nitric oxide produced by human patients undergoing surgical treatment for contusional brain injuries. METHODS Contused brain tissue specimens from eight consecutive patients who underwent surgical treatment for brain contusions 3 hours to 5 days after trauma were evaluated in immunohistochemical analyses. Double-staining assays were used to define which cells produced the different isoforms. RESULTS Increases in iNOS-positive cells were detectable within 6 hours after trauma, with a peak at 8 to 23 hours. Expression of iNOS after trauma was detected in neurons, macrophages, neutrophils, astrocytes, and oligodendrocytes. The cellular sources of iNOS differed at different times after trauma. No detectable difference in the expression of the neuronal or endothelial isoforms was observed for trauma patients, compared with control subjects. CONCLUSION iNOS expression was up-regulated in a time-dependent manner in human brain tissue obtained from patients undergoing surgical treatment for contusional trauma. Our human data largely parallel experimental findings in rats, indicating that such trauma models are relevant for experimental studies and treatment trials.

Reduced neuronal injury after treatment with NG-nitro-L-arginine methyl ester (L-NAME) or 2-sulfo-phenyl-N-tert-butyl nitrone (S-PBN) following experimental brain contusion.

OBJECTIVE: Nitric oxide (NO) and oxygen free radicals are implicated in the pathophysiology of traumatic brain injury (TBI). Peroxynitrite formation from NO and superoxide contributes to secondary neuronal injury but the neuroprotective effects of nitric oxide synthase (NOS)-inhibitors have been contradictory. This study was undertaken to examine whether PTtic administration of the (NOS)-inhibitor NG-nitro-l-arginine methyl ester (L-NAME), and a combination of L-NAME and the nitrone radical scavenger 2-sulfo-phenyl-N-tert-butyl nitrone (S-PBN) favorable affects neuronal injury in a model of TBI. METHODS: A weight-drop model of TBI was used. The animals received L-NAME, S-PBN or a combination of the drugs 15 minutes prothrombin time (PT) and sacrificed after 24 hours or six days. NOS activity was measured by the conversion of L-[U-14C]arginine to L-[U-14C]citrulline. Peroxynitrite formation, cellular apoptosis, neuronal degeneration and survival were assessed by nitrotyrosine-, TUNEL-, Fluoro-Jade- and NeuN–stainings. RESULTS: eNOS and nNOS activity was significantly reduced in animals that received L-NAME alone or the combination with S-PBN. iNOS activity or iNOS immunoreactivity was not affected. All treatments significantly reduced neuronal degeneration and nitrotyrosine immunoreactivity at 24 hours and increased neuronal survival at six days PT. No differences were detected between L-NAME and L-NAME + S-PBN groups. CONCLUSION: NO from NOS contributes to secondary neuronal injury in this TBI-model. PTtic treatment does not inhibit early beneficial NO-related effects. L-NAME and S-PBN limit peroxynitrite formation, promoting neuronal survival. The combination of L-NAME and S-PBN was neuroprotective; surprisingly no additive effects were found on nitrotyrosine formation, apoptosis or neuronal survival.

Neuronal degeneration and iNOS expression in experimental brain contusion following treatment with colchicine, dexamethasone, tirilazad mesylate and nimodipine

SummaryBackground. The pathophysiological mechanisms of secondary neurological injury after traumatic brain injury are complex. Post-traumatic biochemical reactions include parenchymal inflammation, free radical production, increased intracellular calcium and lipid peroxidation and nitric oxide production. The relative importance of each mechanism is unknown in brain contusions. This study was undertaken to investigate protection by the neuroprotective and/or anti-inflammatory drugs that have different putative mechanisms of action: colchicine, dexamethasone, tirilazad mesylate and nimodipine.Method. A brain contusion was produced using a weight-drop model in rats. The animals were treated with either one of the drugs at previously defined relevant dosage or control. Fluoro-Jade labelling, TUNEL-staining and immunohisto-chemistry were used to study neuronal degeneration, cellular apoptosis and iNOS expression. In addition, the number of surviving neurons after 14 days was determined.Findings. The number of degenerating neurons was significantly reduced in all treatment groups at 24 hours while the total number of apoptotic cells including inflammatory cells and glia was unchanged. iNOS-expression was reduced in all treatment groups at 24 hours but not later. Only colchicine and tirilazad mesylate significantly enhanced neuronal survival at 14 days after injury.Conclusions. The findings underscored that an early neuroprotective effect does not necessarily lead to increased long-term neuronal survival. The absence of a significant long-term effect with nimodipine and dexamethasone agrees with clinical studies. Colchicine with an anti-macrophage/anti-inflammatory activity and the free radical scavenger tirilazad mesylate were effective for amelioration of experimental contusion with moderate energy transfer. Early neuroprotection may to some extent target iNOS via different pathways since all tested drugs affected both iNOS expression and neuronal degeneration.

iNOS-mediated secondary inflammatory response differs between rat strains following experimental brain contusion

BackgroundNitric oxide is a key mediator of post-traumatic inflammation in the brain. We examined the expressions of iNOS, nNOS, and eNOS in inbred DA and PVGa rat strains where DA is susceptible to autoimmune neuroinflammation and PVGa-resistant.MethodsParietal contusions using a weight drop model were produced in five rats per genotype. After 24 h, the brains were removed and analyzed using a range of immunohistochemical methods.ResultsPVGa presented significantly increased iNOS expression in infiltrating inflammatory cells in the perilesional area compared to DA (p < 0.05). The amount of w3/13-positive infiltrating inflammatory cells did not differ between strains. eNOS and nNOS expression did not differ between strains. iNOS-positive cells coexpressed neuronal (NeuN), macrophage (ED-1), and leucocyte (w3/13) markers. MnSOD was significantly increased in PVGa (p < 0.05). 3-Nitrotyrosine, a measure of peroxynitrite levels, and fluoro-jade stained neuronal degeneration, did not differ between strains.ConclusionsTwo inbred rat strains with genetically determined differences in susceptibility to develop autoimmune disease displayed different levels of the inflammatory and anti-inflammatory mediators iNOS and MnSOD, indicating genetic regulation. Interestingly, the increased levels of iNOS did not lead to elevated expression of the neuronal cell-death marker fluoro-jade. The increased iNOS expression was correlated with increased expression of superoxide scavenger MnSOD. Excessive peroxynitrite formation was probably prevented by limitation of available superoxide. Subsequently, the higher expression of potentially deleterious iNOS in PVGa did not result in increased neuronal death.

Immunohistochemical localization of OCT2 in the cochlea of various species.

To locate the organic cation transporter 2 (OCT2) in the cochlea of three different species and to modulate the ototoxicity of cisplatin in the guinea pig by pretreatment with phenformin, having a known affinity for OCT2.

Improved Head and Neck Free Flap Outcome—Effects of a Treatment Protocol Adjustment from Pre- to Postoperative Radiotherapy

Background: The impact of preoperative radiotherapy on microvascular reconstructive surgery outcome has been a subject of debate. However, data are conflicting and often dependent on local treatment protocols. We have studied the effects of radiotherapy in a unique, single-center setting where a treatment protocol change was undertaken from pre- to postoperative radiotherapy administration for microsurgical head and neck reconstructions. Methods: A cohort study was conducted for 200 consecutive head and neck free flap cases, where 100 were operated on before and 100 after the treatment protocol adjustment in 2006. Only direct cancer reconstructions were included. Complication rates of anastomosis-related (flap necrosis) and flap bed–related (infection, fistula, and wound dehiscence) complications were compared between irradiated and nonirradiated patients. A multivariate analysis was performed to correct for treatment period. Results: One hundred twenty-six patients had received radiotherapy before reconstruction due to cases of cancer recurrence. There were no significant differences in demographic data or risk factors between irradiated and nonirradiated cases. Irradiated cases had a higher rate of both flap loss (9.5% versus 1.4%; P = 0.034) and flap bed–related complications (29% versus 13%; P = 0.014). However, after multivariate analysis, there was only a significant relationship between preoperative irradiation and infection (odds ratio = 2.51; P = 0.033) and fistula formation (odds ratio = 3.13; P = 0.034). Conclusions: The current single-center study clearly indicates that preoperative radiotherapy is a risk factor for both infection and fistula formation, most likely related to an impaired flap bed. We suggest postoperative radiotherapy administration whenever possible for oncological reasons, otherwise proper antibiotic cover and meticulous flap insetting to prevent radiation-related infection and fistula formation.

Upregulation of Plasminogen Activator Inhibitor-1 in Irradiated Recipient Arteries and Veins from Free Tissue Transfer Reconstruction in Cancer Patients

Background Clinical studies have shown that radiotherapy can induce vascular disease at the site of exposure but is usually not clinically evident until years after treatment. We have studied irradiated human arteries and veins to better understand the underlying biology in search of future treatments. The aim was to investigate whether radiotherapy contributed to a sustained expression of plasminogen activator inhibitor-1 (PAI-1) in human arteries and veins. Methods Irradiated arteries and veins were harvested, together with unirradiated control vessels, from patients undergoing free tissue transfer reconstruction at a median time of 90 weeks [5–650] following radiation exposure. Differential gene expression of PAI-1 was analysed, together with immunohistochemistry (IHC) and immunofluorescence (IF). Results PAI-1 gene expression was increased in both arteries (p = 0.012) and veins (p < 0.001) in irradiated compared to unirradiated control vessels. IHC and IF indicated that cells expressing PAI-1 were located in the adventitia of both arteries and veins and colocalized with cells positive for CD68, CD45, and α-SMA in arteries and with CD45 and α-SMA in veins. Conclusion The current study shows a sustained upregulation of PAI-1 in both arteries and veins after exposure to ionizing radiation, indicating a chronic inflammation mainly in the adventitia. We believe that the results contribute to further understanding of radiation-induced vascular disease, where targeting PAI-1 may be a potential treatment.

No evidence for human papillomavirus having a causal role in salivary gland tumors

BackgroundSalivary gland malignancies are a very heterogeneous group of cancers, with histologically > 20 different subtypes, and prognosis varies greatly. Their etiology is unknown, however, a few small studies show presence of human papillomavirus (HPV) in some subtypes, although the evidence for HPV having a causal role is weak. The aim of this study was to investigate if HPV plays a causal role in the development of different parotid salivary gland tumor subtypes.MethodsDNA was extracted from 107 parotid salivary gland formalin fixed paraffin embedded tumors and 10 corresponding metastases, and tested for 27 different HPV types using a multiplex bead based assay. HPV DNA positive tumors were stained for p16INK4a overexpression by immunohistochemistry.ResultsOne of the 107 malignant parotid salivary gland tumors (0.93%) and its corresponding metastasis on the neck were positive for HPV16 DNA, and both also overexpressed p16INK4a. The HPV positive primary tumor was a squamous cell carcinoma; neither mucoepidermoid nor adenoid cystic tumors were found HPV positive.ConclusionsIn conclusion, HPV DNA analysis in a large number of malignant parotid salivary gland tumors, including 12 different subtypes, did not show any strong indications that tested HPV types have a causal role in the studied salivary gland tumor types.