Histopathology of traumatic brain injury to the developing brain

Trauma to the developing brain leads to necrotic lesion at the site of injury and delayed apoptotic neurodegeneration at distant sites mainly in the thalamus, caudate nucleus and cortex. We studied the distribution and timely activation of monocytes/macrophages, microglia, astrocytes and two inflammatory cytokines, interleukin (IL)-1ß and IL-18, 2 h to 14 days following trauma using biochemistry and immunohistochemistry. A marked increase of mRNA and protein levels for IL-1ß and IL-18 was detected 2-12 h after injury. Apoptotic cell death affects mostly neuronal populations ipsilateral to the injury 6 h to 5 days later. Microglial activation was first evident at 12 h, peaked at 36-48 h and decreased substantially by 5 days. Astrocytic activation started at 18 h, peaked at 48 h and gradually declined by 14 days after trauma. The activation of immune and glial cells together with increased expression of both interleukins occurred at the site of primary and secondary damages. Our findings suggest that reactive microglia/astrocytes at the sites of secondary lesions might maintain apoptotic neurodegeneration over several days after traumatic injury to the immature brain but they might also promote tissue repair. Understanding the role of glial cells to progression of inflammation and apoptotic neurodegeneration together with tissue repair in the developing brain may provide valuable information to guide treatment after brain trauma

Neuroinflammation after traumatic injury to the developing brain

PURPOSES: Mechanical trauma to the developing rodent brain induces a diffuse secondary neuroapoptosis associated with infiltration of immune cells, local and systemic increased levels of proinflammatory mediators. Our aim was to study their expression, cellular localization, distribution pattern and time course in various brain regions. MATERIALS AND METHODS: 7-day-old Wistar rats and C57/BL6 mice were subjected to cortical trauma. Animals were sacrificed at defined time points - from 2 h to 14 days following trauma. Brain tissues were processed for molecular analyses, single or double indirect peroxidase/fluorescence immunohistochemistry for apoptotic cell death, microglia and interleukin (IL)-1ß/IL-18. RESULTS: Apoptotic neuronal cell death detected by TUNEL was found at distant regions to trauma site mainly ipsilateral from 6 h to 5 days later. A substantial activation of ED1+ microglia occurred at the site of primary and secondary damages. It was first evident at 12 h, peaked at 36-48 h and decreased significantly after 5 days. A marked increase of mRNA, protein levels and imunohistochemical expression of two pro-inflammatory cytokines, interleukin (IL)-1ß and IL-18, was found from 2 h to 5 days following trauma. Mice deficient in IL-18 (IL-18−/−) were protected against post-traumatic brain damage. CONCLUSIONS: Brain trauma leads to neuroinflammation expressed by microglial activation and an increase in IL-1ß and IL-18. Activated microglia are one of the main cellular sources of elevated levels for both cytokines. They are probably involved in and help sustain apoptotic neurodegeneration over several days after trauma. This finding might define microglia and IL-1ß/IL-18 as potential post-traumatic therapeutic targets

Dexmedetomidine Prevents Lipopolysaccharide-Induced MicroRNA Expression in the Adult Rat Brain

During surgery or infection, peripheral inflammation can lead to neuroinflammation, which is associated with cognitive impairment, neurodegeneration, and several neurodegenerative diseases. Dexmedetomidine, an α-2-adrenoceptor agonist, is known to exert anti-inflammatory and neuroprotective properties and reduces the incidence of postoperative cognitive impairments. However, on the whole the molecular mechanisms are poorly understood. This study aims to explore whether dexmedetomidine influences microRNAs (miRNAs) in a rat model of lipopolysaccharide (LPS)-induced neuroinflammation. Adult Wistar rats were injected with 1 mg/kg LPS intraperitoneal (i.p.) in the presence or absence of 5 µg/kg dexmedetomidine. After 6 h, 24 h, and 7 days, gene expressions of interleukin 1-β (IL1-β), tumor necrosis factor-α (TNF-α), and microRNA expressions of miR 124, 132, 134, and 155 were measured in the hippocampus, cortex, and plasma. Dexmedetomidine decreased the LPS-induced neuroinflammation in the hippocampus and cortex via significant reduction of the IL1-β and TNF-α gene expressions after 24 h. Moreover, the LPS-mediated increased expressions of miR 124, 132, 134, and 155 were significantly decreased after dexmedetomidine treatment in both brain regions. In plasma, dexmedetomidine significantly reduced LPS- induced miR 155 after 6 h. Furthermore, there is evidence that miR 132 and 134 may be suitable as potential biomarkers for the detection of neuroinflammation. View Full-Tex

Dexmedetomidine Restores Autophagic Flux, Modulates Associated microRNAs and the Cholinergic Anti-inflammatory Pathway upon LPS-Treatment in Rats

Infections and perioperative stress can lead to neuroinflammation, which in turn is linked to cognitive impairments such as postoperative delirium or postoperative cognitive dysfunctions. The alpha 2-adrenoceptor agonist dexmedetomidine (DEX) prevents cognitive impairments and has organo-protective and anti-inflammatory properties. Macroautophagy (autophagy) regulates many biological processes, but its role in DEX-mediated anti-inflammation and the underlying mechanism of DEX remains largely unclear. We were interested how a pretreatment with DEX protects against lipopolysaccharide (LPS)-induced inflammation in adult male Wistar rats. We used Western blot and activity assays to study how DEX modulated autophagy- and apoptosis-associated proteins as well as molecules of the cholinergic anti-inflammatory pathway, and qPCR to analyse the expression of autophagy and inflammation-associated microRNAs (miRNA) in the spleen, cortex and hippocampus at different time points (6 h, 24 h, 7 d). We showed that a DEX pretreatment prevents LPS-induced impairments in autophagic flux and attenuates the LPS-induced increase in the apoptosis-associated protein cleaved poly(ADP-ribose)-polymerase (PARP) in the spleen. Both, DEX and LPS altered miRNA expression and molecules of the cholinergic anti-inflammatory pathway in the spleen and brain. While only a certain set of miRNAs was up- and/or downregulated by LPS in each tissue, which was prevented or attenuated by a DEX pretreatment in the spleen and hippocampus, all miRNAs were up- and/or downregulated by DEX itself - independent of whether or not they were altered by LPS. Our results indicate that the organo-protective effect of DEX may be mediated by autophagy, possibly by acting on associated miRNAs, and the cholinergic anti-inflammatory pathway

A Modified Approach to Induce Predictable Congestive Heart Failure by Volume Overload in Rats

The model of infrarenal aortocaval fistula (ACF) has recently gained new interest in its use to investigate cardiac pathophysiology. Since in previous investigations the development of congestive heart failure (CHF) was inconsistent and started to develop earliest 8-10 weeks after fistula induction using a 18G needle, this project aimed to induce a predictable degree of CHF within a definite time period using a modified approach. An aortocaval fistula was induced in male Wistar rats using a 16G needle as a modification of the former 18G needle-technique described by Garcia and Diebold. Results revealed within 28 +/- 2 days of ACF significantly increased heart and lung weight indices in the ACF group accompanied by elevated filling pressure. All hemodynamic parameters derived from a pressure-volume conductance-catheter in vivo were significantly altered in the ACF consistent with severe systolic and diastolic left ventricular dysfunction. This was accompanied by systemic neurohumoral activation as demonstrated by elevated rBNP-45 plasma concentrations in every rat of the ACF group. Furthermore, the restriction in overall cardiac function was associated with a beta 1- and beta 2-adrenoreceptor mRNA downregulation in the left ventricle. In contrast, beta 3-adrenoreceptor mRNA was upregulated. Finally, electron microscopy of the left ventricle of rats in the ACF group showed signs of progressive subcellular myocardial fragmentation. In conclusion, the morphometric, hemodynamic and neurohumoral characterization of the modified approach revealed predictable and consistent signs of congestive heart failure within 28 +/- 2 days. Therefore, this modified approach might facilitate the examination of various questions specific to CHF and allow for pharmacological interventions to determine pathophysiological pathways

Effects of repetitive exposure to pain and morphine treatment on the neonatal rat brain

Background: Untreated exposure to pain in preterm neonates might damage the vulnerable premature brain and alter development. Pain treatment is limited because analgesic agents may also have adverse neurodevelopmental consequences in newborns. Objective: To study the effects of neonatal pain and morphine treatment on the developing brain in a n

Synaptic NMDA receptor activity boosts intrinsic antioxidant defenses

Intrinsic antioxidant defenses are important for neuronal longevity. We found that in rat neurons, synaptic activity, acting via NMDA receptor (NMDAR) signaling, boosted antioxidant defenses by making changes to the thioredoxin-peroxiredoxin (Prx) system. Synaptic activity enhanced thioredoxin activity, facilitated the reduction of overoxidized Prxs and promoted resistance to oxidative stress. Resistance was mediated by coordinated transcriptional changes; synaptic NMDAR activity inactivated a previously unknown Forkhead box O target gene, the thioredoxin inhibitor Txnip. Conversely, NMDAR blockade upregulated Txnip in vivo and in vitro, where it bound thioredoxin and promoted vulnerability to oxidative damage. Synaptic activity also upregulated the Prx reactivating genes Sesn2 (sestrin 2) and Srxn1 (sulfiredoxin), via C/EBPβ and AP-1, respectively. Mimicking these expression changes was sufficient to strengthen antioxidant defenses. Trans-synaptic stimulation of synaptic NMDARs was crucial for boosting antioxidant defenses; chronic bath activation of all (synaptic and extrasynaptic) NMDARs induced no antioxidative effects. Thus, synaptic NMDAR activity may influence the progression of pathological processes associated with oxidative damage

Molecular mechanisms of hyperoxia- and trauma-induced brain injury in the developing brain of mouse and rat

Trotz medizinischer Fortschritte geht die sinkende Mortalität von Frühgeborenen weiter mit einer hohen Rate neurologischer Defizite einher. In der Behandlung von kranken Früh- und Neugeborenen ist Sauerstoff ein bedeutendes Therapeutikum, wobei die Effekte einer Hyperoxie auf das neonatale Gehirn zunehmend das Interesse der Wissenschaft wecken, da diese für entstehende neuronale Schädigungen mitverantwortlich gemacht werden. Die traumatische Hirnschädigung ist ein wesentlicher Grund für den Tod und neurologische Residualsyndrome in der Kindheit. Im Gegensatz zum Trauma des Erwachsenen, sind die dem kindlichen Hirntrauma zugrundeliegenden Pathomechanismen noch weitgehend ungeklärt. Im Rahmen der vorliegenden Arbeit wurden neurodegenerative Schädigungen von Hyperoxie bzw. Schädelhirntrauma auf das sich entwickelnde Gehirn von Maus und Ratte histologisch nachgewiesen, wobei sich Neurodegenerationen in verschiedenen Hirnregionen zeigten. Es kam jeweils zu einer Aktivierung von neuroinflammatorischen Prozessen mit einem Anstieg von Caspase-1 und den von Caspase-1 aktivierten proinflammatorischen Zytokinen Interleukin-1ß (IL-1ß) und Interleukin-18 (IL-18). Die intraperitoneale (i.p.) Gabe von rekombinantem Interleukin-18-Bindungsprotein (IL-18BP) führte zu einer Reduzierung des apoptotischen Zelltodes in beiden Schädigungsmodellen. Zudem waren IL-1 receptor-associated kinase-4 (IRAK-4)-defiziente Mäuse weitgehend geschützt gegen den Hyperoxie-induzierten Zelltod und IL-18-defiziente Mäuse gegen den Zelltod nach Schädelhirntrauma. Nach Schädelhirntrauma wurden zusätzlich die beiden Matrixmetalloproteinasen (MMPs) MMP-2 und -9 auf Gen- und Proteinebene hochreguliert und deren Inhibitoren tissue inhibitors of metalloproteinases (TIMPs) TIMP-1 und TIMP-2 in der Expression reduziert. Die i.p. Applikation des MMP-Inhibitors GM6001 2 Stunden nach Schädelhirntrauma führte zu einer Verringerung der apoptotischen Neurodegeneration und stellt somit eine mögliche therapeutische Anwendung nach Schädelhirntrauma dar. Im Hyperoxiemodell führte die gleichzeitige i.p. Applikation von rekombinantem Erythropoietin (rEPO) zu einer Abnahme des apoptotischen Zelltodes und des oxidativen Stresses nach Hyperoxie. Der Hyperoxie-induzierte Aktivitätsanstieg von Caspase-2, -3 und -8 wurde durch rEPO-Gabe reduziert. Außerdem wurde der durch Hyperoxie verminderte Gehalt des neurotrophen Faktors brain-derived neurotrophic factor (BDNF) sowie der phosphorylierten, aktiven Formen der Kinasen Akt (Proteinkinase B) und ERK1/2 durch Gabe von rEPO wieder erhöht, so dass die neuroprotektive Eigenschaft von rEPO im Modell der Sauerstofftoxizität und daran beteiligte Mechanismen nachgewiesen werden konnten.Although the mortality of infants with extremly low birth weight and gestational age decreased markedly because of advances in medical care, long- term neurological deficits continue in a high rate. Oxygen is an important therapeutic agent for the treatment of preterm and term infants whereupon hyperoxic effects to the immature brain awake an increased scientific opinion, because they are responsible for neuronal damages. Traumatic brain injury constitutes a major cause of morbidity and neurological residual syndromes in children. In contrast to trauma in adults the pathophysiology of traumatic brain injury to the developing brain is widely unknown. In this present work hyperoxic and traumatic neurodegenerative damages to different regions of the developing brain of mouse and rat were shown by histology. There was an activation of neuroinflammatory processes with an increase of caspase-1 and the caspase-1-activated proinflammatory cytokines interleukin-1ß (IL-1ß) and interleukin-18 (IL-18). Intraperitoneal injection of recombinant interleukin-18-binding protein (IL-18BP) led to a reduction of apoptotic cell death in both injury models. In addition mice deficient in IL-1 receptor- associated kinase-4 (IRAK-4) were widely protected against hyperoxia-induced cell death and mice deficient in IL-18 were protected against cell death after traumatic brain injury. Furthermore matrix metalloproteinases (MMPs) MMP-2 and -9 were upregulated on mRNA and protein level after traumatic brain injury, whereas expression of the tissue inhibitors of metalloproteinases (TIMPs) TIMP-1 and TIMP-2 was reduced. Intraperitoneal injection of GM6001 (Ilomastat), an MMP inhibitor, 2 h after trauma, substantially attenuated apoptotic neurodegeneration and might serve as a therapeutic approach after traumatic brain injury. A decrease of apoptotic cell death and oxidative stress was detected when recombinant erythropoietin (rEPO) was intraperitoneal injected concomitant with the start of hyperoxic conditions. Treatment with rEPO reduces the hyperoxia-induced increase of caspase-2, -3 and -8 activities. Moreover hyperoxia-reduced levels of brain-derived neurotrophic factor (BDNF), phosphorylated, active pAkt (proteinkinase B) and pERK1/2 were ameliorated through rEPO administration. Hence the neuroprotective feature of rEPO in the model of oxygen toxicity was shown and mechanisms which are involved in this context