Illustrating cerebral function: the iconography of arrows

For over a century the arrow has appeared in illustrations of cerebral function, yet the implications of using such symbols have not been previously considered. This review seeks to outline the nature, evolution, applications and limitations of this deceptively simple graphic device when it is used to picture functions of the brain. The arrow is found to have been used in several different ways: as a means of endowing anatomical structures with functional properties; as a method of displaying neural function either in free-standing form or in a structural or spatial framework; as a device for correlating functional data with underlying brain topography; and as a technique for linking functions of the brain with the world outside and with various philosophical concepts. For many of these uses the essential feature of the arrow is its directional characteristic. In contrast to the line, it is direction that enables the arrow to display information about time, which in turn can be exploited to depict functional rather than structural data. However, the use of the arrow is fraught with difficulties. It is often unclear whether an arrow has been used to illustrate fact, hypothesis, impression or possibility, or merely to provide a decorative flourish. Furthermore, the powerful symbolic nature of the arrow can so easily confer a spurious validity on the conjectural. Increasingly now there are insuperable difficulties when attempting to illustrate complex mechanisms of brain function. In the iconography of cerebral function, therefore, arrows with all their ambiguities may in certain circumstances become superseded by more non-representational symbols such as the abstract devices of the computational neuroscientist.     

The heart-brain connection: mechanistic insights and models

While both cardiac dysfunction and progressive loss of cognitive function are prominent features of an ageing population, surprisingly few studies have addressed the link between the function of the heart and brain. Recent literature indicates that autoregulation of cerebral flow is not able to protect the brain from hypoperfusion when cardiac output is reduced or atherosclerosis is prominent. This suggests a close link between cardiac function and large vessel atherosclerosis on the one hand and brain perfusion and cognitive functioning on the other. Mechanistically, the presence of vascular pathology leads to chronic cerebral hypoperfusion, blood brain barrier breakdown and inflammation that most likely precede neuronal death and neurodegeneration. Animal models to study the effects of chronic cerebral hypoperfusion are available, but they have not yet been combined with cardiovascular models.     

Melatonin reverses pinealectomy-induced decrease of benzodiazepine binding in rat cerebral cortex

Pinealectomy of rats resulted in significant depression of benzodiazepine receptors (assessed by [³H]flunitrazepam binding) in cerebral cortex 3–14 days after surgery without affecting their affinity significantly. A single s.c. injection of melatonin (800 μg/kg body wt) restored the depressed brain benzodiazepine receptor sites. Single melatonin injections (up to 1600 μg/kg) to intact rats did not affect brain benzodiazepine binding when injected at either morning or evening hours. Daily melatonin treatment to intact rats for 5 days augmented benzodiazepine receptor density in brain (morning injections) or its dissociation constant (evening injections). Melatonin added in vitro to rat cerebral cortex membranes only slightly depressed [³H]flunitrazepam binding at 100 μM concentrations. These results point out a link between pineal activity and benzodiazepine receptor function in rats. They also indicate that pharmacological doses of melatonin affect benzodiazepine binding sites in rat cerebral cortex.     

Lack of desensitization of muscarinic receptor-mediated second messenger signals in rat brain upon acute and chronic inhibition of acetylcholinesterase.

We studied the effects of acute and chronic in vivo inhibition of acetylcholinesterase on both the density and function of brain muscarinic cholinergic receptors. Adult male rats were treated either once or multiple times over a period of 10 days with the irreversible acetylcholinesterase inhibitor diisopropylfluorophosphate (DFP). The concentration and affinity of muscarinic receptors in various brain regions were determined using radioligand binding techniques. Acute DFP treatment resulted in a significant reduction in receptor number only in the brain stem, while chronic treatment caused receptor down-regulation in the brain stem, cerebral cortex, and striatum. There was no change in ligand affinity in any of the brain regions. In sharp contrast, muscarinic receptor function was fully preserved, in terms of coupling of the receptors to increased phosphoinositide hydrolysis in the cerebral cortex, hippocampus, and striatum, or inhibition of cyclic AMP formation in the cerebral cortex or striatum. Therefore, there is a marked lack or correlation between DFP-induced muscarinic receptor down-regulation and receptor desensitization.     

Lateralized behaviour of a non-social cichlid fish (Amatitlania nigrofasciata) in a social and a non-social environment

Cerebral lateralization, the partitioning of cognitive function preferentially into one hemisphere of the brain, is a trait ubiquitous among vertebrates. Some species exhibit population level lateralization, where the pattern of cerebral lateralization is the same for most members of that species; however, other species show only individual level lateralization, where each member of the species has a unique pattern of lateralized brain function. The pattern of cerebral lateralization within a population and an individual has been shown to differ based on the stimulus being processed. It has been hypothesized that sociality within a species, such as shoaling behaviour in fish, may have led to the development and persistence of population level lateralization. Here we assessed cerebral lateralization in convict cichlids (Amatitlania nigrofasciata), a species that does not shoal as adults but that shoals briefly as juveniles. We show that both male and female convict cichlids display population level lateralization when in a solitary environment but only females show population level lateralization when in a perceived social environment. We also show that the pattern of lateralization differs between these two tasks and that strength of lateralization in one task is not predictive of strength of lateralization in the other task.     

Lack of desensitization of muscarinic receptor–mediated second messenger signals in rat brain upon acute and chronic inhibition of acetylcholinesterase

We studied the effects of acute and chronic in vivo inhibition of acetylcholinesterase on both the density and function of brain muscarinic cholinergic receptors. Adult male rats were treated either once or multiple times over a period of 10 days with the irreversible acetylcholinesterase inhibitor diisopropylfluorophosphate (DFP). The concentration and affinity of muscarinic receptors in various brain regions were determined using radioligand binding techniques. Acute DFP treatment resulted in a significant reduction in receptor number only in the brain stem, while chronic treatment caused receptor downregulation in the brain stem, cerebral cortex, and striatum. There was no change in ligand affinity in any of the brain regions. In sharp contrast, muscarinic receptor function was fully preserved, in terms of coupling of the receptors to increased phosphoinositide hydrolysis in the cerebral cortex, hippocampus, and striatum, or inhibition of cyclic AMP formation in the cerebral cortex or striatum. Therefore, there is a marked lack or correlation between DFP-induced muscarinic receptor down-regulation and receptor desensitization.     

Determination of Brain Death

With the careful application of the principles outlined herein, brain death can be determined with certainty. There have been no documented reports of survivors when these guidelines have been followed. The traid of a known mechanism of brain injury, absence of contributing metabolic or toxic central nervous system depression and absence of demonstrable brain function is sufficient to determine brain death clinically and, in most states, legally. The use of apneic oxygenation protects cadaver organs for transplantation during the period needed to prove that a patient cannot breathe.Very little can ameliorate the tragedy of sudden and unexpected fatal cerebral injury. Nonetheless, the concept of brain death is well established, and there is no longer a medical or an ethical reason to prolong unnecessary support of these patients.     

Delayed astrocytic contact with cerebral blood vessels in FGF‐2 deficient mice does not compromise permeability properties at the developing blood‐brain barrier

The brain functions within a specialized environment tightly controlled by brain barrier mechanisms. Understanding the regulation of barrier formation is important for understanding brain development and may also lead to finding new ways to deliver pharmacotherapies to the brain; access of many potentially promising drugs is severely hindered by these barrier mechanisms. The cellular composition of the neurovascular unit of the blood‐brain barrier proper and their effects on regulation of its function are beginning to be understood. One hallmark of the neurovascular unit in the adult is the astroglial foot processes that tightly surround cerebral blood vessels. However their role in barrier formation is still unclear. In this study we examined barrier function in newborn, juvenile and adult mice lacking fibroblast growth factor‐2 (FGF‐2), which has been shown to result in altered astroglial differentiation during development. We show that during development of FGF‐2 deficient mice the astroglial contacts with cerebral blood vessels are delayed compared with wild‐type animals. However, this delay did not result in changes to the permeability properties of the blood brain barrier as assessed by exclusion of either small or larger sized molecules at this interface. In addition cerebral vessels were positive for tight‐junction proteins and we observed no difference in the ultrastructure of the tight‐junctions. The results indicate that the direct contact of astroglia processes to cerebral blood vessels is not necessary for either the formation of the tight‐junctions or for basic permeability properties and function of the blood‐brain barrier. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1201–1212, 2016     

Single Sustained Inflation followed by Ventilation Leads to Rapid Cardiorespiratory Recovery but Causes Cerebral Vascular Leakage in Asphyxiated Near-Term Lambs

BackgroundA sustained inflation (SI) rapidly restores cardiac function in asphyxic, bradycardic newborns but its effects on cerebral haemodynamics and brain injury are unknown. We determined the effect of different SI strategies on carotid blood flow (CaBF) and cerebral vascular integrity in asphyxiated near-term lambs.MethodsLambs were instrumented and delivered at 139 ± 2 d gestation and asphyxia was induced by delaying ventilation onset. Lambs were randomised to receive 5 consecutive 3 s SI (multiple SI; n = 6), a single 30 s SI (single SI; n = 6) or conventional ventilation (no SI; n = 6). Ventilation continued for 30 min in all lambs while CaBF and respiratory function parameters were recorded. Brains were assessed for gross histopathology and vascular leakage.ResultsCaBF increased more rapidly and to a greater extent during a single SI (p = 0.01), which then decreased below both other groups by 10 min, due to a higher cerebral oxygen delivery (p = 0.01). Blood brain barrier disruption was increased in single SI lambs as indicated by increased numbers of blood vessel profiles with plasma protein extravasation (p = 0.001) in the cerebral cortex. There were no differences in CaBF or cerebral oxygen delivery between the multiple SI and no SI lambs.ConclusionsVentilation with an initial single 30 s SI improves circulatory recovery, but is associated with greater disruption of blood brain barrier function, which may exacerbate brain injury suffered by asphyxiated newborns. This injury may occur as a direct result of the initial SI or to the higher tidal volumes delivered during subsequent ventilation.     

A Type-II Positive Allosteric Modulator of α7 nAChRs Reduces Brain Injury and Improves Neurological Function after Focal Cerebral Ischemia in Rats

In the absence of clinically-efficacious therapies for ischemic stroke there is a critical need for development of new therapeutic concepts and approaches for prevention of brain injury secondary to cerebral ischemia. This study tests the hypothesis that administration of PNU-120596, a type-II positive allosteric modulator (PAM-II) of α7 nicotinic acetylcholine receptors (nAChRs), as long as 6 hours after the onset of focal cerebral ischemia significantly reduces brain injury and neurological deficits in an animal model of ischemic stroke. Focal cerebral ischemia was induced by a transient (90 min) middle cerebral artery occlusion (MCAO). Animals were then subdivided into two groups and injected intravenously (i.v.) 6 hours post-MCAO with either 1 mg/kg PNU-120596 (treated group) or vehicle only (untreated group). Measurements of cerebral infarct volumes and neurological behavioral tests were performed 24 hrs post-MCAO. PNU-120596 significantly reduced cerebral infarct volume and improved neurological function as evidenced by the results of Bederson, rolling cylinder and ladder rung walking tests. These results forecast a high therapeutic potential for PAMs-II as effective recruiters and activators of endogenous α7 nAChR-dependent cholinergic pathways to reduce brain injury and improve neurological function after cerebral ischemic stroke.     

Iron overload induces cerebral endothelial senescence in aged mice and in primary culture in a sex-dependent manner

Iron imbalance in the brain negatively affects brain function. With aging, iron levels increase in the brain and contribute to brain damage and neurological disorders. Changes in the cerebral vasculature with aging may enhance iron entry into the brain parenchyma, leading to iron overload and its deleterious consequences. Endothelial senescence has emerged as an important contributor to age-related changes in the cerebral vasculature. Evidence indicates that iron overload may induce senescence in cultured cell lines. Importantly, cells derived from female human and mice generally show enhanced senescence-associated phenotype, compared with males. Thus, we hypothesize that cerebral endothelial cells (CEC) derived from aged female mice are more susceptible to iron-induced senescence, compared with CEC from aged males. We found that aged female mice, but not males, showed cognitive deficits when chronically treated with ferric citrate (FC), and their brains and the brain vasculature showed senescence-associated phenotype. We also found that primary culture of CEC derived from aged female mice, but not male-derived CEC, exhibited senescence-associated phenotype when treated with FC. We identified that the transmembrane receptor Robo4 was downregulated in the brain vasculature and in cultured primary CEC derived from aged female mice, compared with those from male mice. We discovered that Robo4 downregulation contributed to enhanced vulnerability to FC-induced senescence. Thus, our study identifies Robo4 downregulation as a driver of senescence induced by iron overload in primary culture of CEC and a potential risk factor of brain vasculature impairment and brain dysfunction.     

Brain and blood indole metabolites after peripheral administration of14C-5-HT in rat

Radioactive techniques were used to reexamine the reports that pharmacological quantities of peripheral serotonin (5-hydroxytryptamine or 5-HT) gain access to brain parenchyma. Intravenous injection of 0.108–4.19 mg/kg of¹⁴C-5-HT (3.55 Ci/100 g weight) produced significant metabolic differences in brain but not blood as a function of dose at up to 10 min after injection. Neither of the metabolites, 5-hydroxyindoleacetic acid nor 5-hydroxytryptophol, were detectable in brain following their intravenous injection, suggesting that when such metabolites are found in brain they represent central metabolism. It has also been shown that peripheral compartments in general, and specifically blood in the cerebral vasculature and the adrenergic nerve endings in the cerebral blood vessels, contribute to the uptake and metabolism of 5-HT. We conclude that doses up to 0.435 mg/kg 5-HT do not cross the blood-brain barrier in the rat but are being totally metabolized in nonneuronal tissues that are invariably removed and assayed along with brain parenchyma. The level at which 5-HT actually passes the blood-brain barrier was found to be at least 0.863 mg/kg. This value is one-third lower than that previously reported.     

Regulation of brain water during acute hyperosmolality in ovine fetuses, lambs, and adults.

In adult rats, when plasma osmolality increases, water flows across the blood-brain barrier down its concentration gradient from brain to plasma, and brain volume deceases. The brain responds to this stress by gaining osmotically active solutes, which limit water loss. This phenomenon is termed brain volume (water) regulation. We tested the hypothesis that brain volume regulation is more effective in young lambs and adult sheep than in fetuses, premature lambs, and newborn lambs. Brain water responses to acute hyperosmolality were measured in the cerebral cortex, cerebellum, and medulla of fetuses at 60 and 90% of gestation, premature ventilated lambs at 90% of gestation, newborn lambs, young lambs at 20-30 days of age, and adult sheep. After exposure of the sheep to increases in systemic osmolality with mannitol plus NaCl, brain water content and electrolytes were quantified. The ideal osmometer is a system in which impermeable solutes do not enter or leave in response to an osmotic stress. There were significant differences from an ideal osmometer in the cerebral cortex of fetuses at 90% of gestation, cerebral cortex, and cerebellum of newborn lambs, and cerebral cortex, cerebellum, and medulla of young lambs and adult sheep; however, there were no differences in the brain regions of fetuses at 60% of gestation and premature lambs, cerebellum and medulla of fetuses at 90% of gestation, and medulla of newborn lambs. We conclude that 1) brain water loss is maximal and brain volume regulation impaired in most brain regions of fetuses at 60 and 90% of gestation and premature lambs; 2) brain volume regulation develops first in the cerebral cortex of the fetuses at 90% of gestation and in the cerebral cortex and cerebellum of newborn lambs, and then it develops in the medulla of the lambs at 20-30 days of age; 3) brain water loss is limited and volume regulation present in the brain regions of young lambs and adult sheep; and 4) the ability of the brain to exhibit volume regulation develops in a region- and age-related fashion.     

Penetration of verapamil across blood brain barrier following cerebral ischemia depending on both paracellular pathway and P-glycoprotein transportation

Background

Blood brain barrier (BBB) dysfunction is a common facet of cerebral ischemia, and the alteration of drug transporter, P-glycoprotein (P-gp), has been documented.

Aims

This study explores influence of damaged BBB and elevated P-gp on cerebral verapamil penetration after ischemia both in vivo and in vitro.

Methods

Middle cerebral artery occlusion (MCAO) induced ischemia/reperfusion (I/R) of rats, and Na₂S₂O₄ induced hypoxia/reoxygenation (H/R) damage of rat brain mirovessel endothelial cells (RBMECs) respectively, served as BBB breakdown model in vivo and in vitro. Evans-Blue (EB) extravagation and ¹²⁵I-albumin were used to quantify BBB dysfunction; UPLC–MS/MS analytical method was performed to determine accurately the concentration of verapamil in brain tissue and cell. Flow cytometry, immunohistochemistry and western blotting were applied to evaluate transport function and protein expression of P-gp.

Results

Overexpressed ICAM-1 and MMP-9 mediated BBB dysfunction after ischemia, which induced EB leakage and ¹²⁵I-albumin uptake increase. Enhanced accumulation of verapamil in brain tissue, but intracellular concentration reduced evidently after H/R injury. Transcellular transportation of verapamil elevated when P-gp function or expression was inhibited after H/R injury.

Conclusion

These data indicated that BBB penetration of verapamil under ischemia condition was not only depending on BBB breakdown, but also regulated by P-gp.     

Neuroprotection: lessons from hibernators

Mammals that hibernate experience extreme metabolic states and body temperatures as they transition between euthermia, a state resembling typical warm blooded mammals, and prolonged torpor, a state of suspended animation where the brain receives as low as 10% of normal cerebral blood flow. Transitions into and out of torpor are more physiologically challenging than the extreme metabolic suppression and cold body temperatures of torpor per se. Mammals that hibernate show unprecedented capacities to tolerate cerebral ischemia, a decrease in blood flow to the brain caused by stroke, cardiac arrest or brain trauma. While cerebral ischemia often leads to death or disability in humans and most other mammals, hibernating mammals suffer no ill effects when blood flow to the brain is dramatically decreased during torpor or experimentally induced during euthermia. These animals, as adults, also display rapid and pronounced synaptic flexibility where synapses retract during torpor and rapidly re-emerge upon arousal. A variety of coordinated adaptations contribute to tolerance of cerebral ischemia in these animals. In this review we discuss adaptations in heterothermic mammals that may suggest novel therapeutic targets and strategies to protect the human brain against cerebral ischemic damage and neurodegenerative disease.     

Simultaneous measurement of glucose transport and utilization in the human brain

Glucose is the primary fuel for brain function, and determining the kinetics of cerebral glucose transport and utilization is critical for quantifying cerebral energy metabolism. The kinetic parameters of cerebral glucose transport, K(M)(t) and V(max)(t), in humans have so far been obtained by measuring steady-state brain glucose levels by proton ((1)H) NMR as a function of plasma glucose levels and fitting steady-state models to these data. Extraction of the kinetic parameters for cerebral glucose transport necessitated assuming a constant cerebral metabolic rate of glucose (CMR(glc)) obtained from other tracer studies, such as (13)C NMR. Here we present new methodology to simultaneously obtain kinetic parameters for glucose transport and utilization in the human brain by fitting both dynamic and steady-state (1)H NMR data with a reversible, non-steady-state Michaelis-Menten model. Dynamic data were obtained by measuring brain and plasma glucose time courses during glucose infusions to raise and maintain plasma concentration at ～17 mmol/l for ～2 h in five healthy volunteers. Steady-state brain vs. plasma glucose concentrations were taken from literature and the steady-state portions of data from the five volunteers. In addition to providing simultaneous measurements of glucose transport and utilization and obviating assumptions for constant CMR(glc), this methodology does not necessitate infusions of expensive or radioactive tracers. Using this new methodology, we found that the maximum transport capacity for glucose through the blood-brain barrier was nearly twofold higher than maximum cerebral glucose utilization. The glucose transport and utilization parameters were consistent with previously published values for human brain.     

Glutamate (mGluR-5) gene expression in brain regions of streptozotocin induced diabetic rats as a function of age: role in regulation of calcium release from the pancreatic islets in vitro

Metabotrophic glutamate receptors (mGluRs) modulate cellular activities involved in the processes of differentiation and degeneration. In this study, we have analysed the expression pattern of group-I metabotropic glutamate receptor (mGlu-5) in cerebral cortex, corpus striatum, brainstem and hippocampus of streptozotocin induced and insulin treated diabetic rats (D+I) as a function of age. Also, the functional role of glutamate receptors in intra cellular calcium release from the pancreatic islets was studied in vitro. The gene expression studies showed that mGlu-5 mRNA in the cerebral cortex increased siginficantly in 7 weeks old diabetic rats whereas decreased expression was observed in brainstem, corpus striatum and hippocampus when compared to control. 90 weeks old diabetic rats showed decreased expression in cerebral cortex, corpus striatum and hippocampus whereas in brainstem the expression increased significantly compared to their respective controls. In 7 weeks old D+I group, mGlu-5 mRNA expression was significantly decreased in cerebral cortex and corpus striatum whereas the expression increased significantly in brainstem and hippocampus. 90 weeks old D+I group showed an increased expression in cerebral cortex, while it was decreased significantly in corpus striatum, brainstem and hippocampus compared to their respective controls. In vitro studies showed that glutamate at lower concentration (10^-7 M) stimulated calcium release from the pancreatic islets. Our results suggest that mGlu-5 receptors have differential expression in brain regions of diabetes and D+I groups as a function of age. This will have clinical significance in management of degeneration in brain function and memory enhancement through glutamate receptors. Also, the regulatory role of glutamate receptors in calcium release has immense therapeutic application in insulin secretion and function.     

Decreased dopamine D2 receptor function in cerebral cortex and brain stem: their role in hepatic ALDH regulation in ethanol treated rats

Ethanol exerts numerous pharmacological effects through its interaction with various neurotransmitters. The dopaminergic pathway is associated with cognitive, endocrine, and motor functions, and reinforcement of addictive substances or behaviours. Aldehyde dehydrogenase (ALDH) is a vital enzyme involved with alcohol metabolism and detoxification. In the present study, we investigated the role of cerebral cortex and brain stem dopamine D₂ receptors in the functional regulation on ALDH enzyme activity, in ethanol administrated rats. Two groups of rats were selected viz. control and alcoholic. Cerebral cortex, brain stem and the liver dopamine content was decreased significantly (P < 0.05, 0.05, 0.001, respectively) and homovanillic acid/dopamine (HVA/DA) ratio has significantly increased (P < 0.05, 0.001 and 0.001), respectively in ethanol treated rats when compared to control. Scatchard analysis of [³H]YM-09151-2 binding to synaptic membrane preparations of cerebral cortex and brain stem showed a significant decrease (P < 0.001, 0.05, respectively) in B _max in ethanol treated rats compared to control and the K _d also decreased significantly (P < 0.05). The ALDH analysis showed a significant increase (P < 0.05) in V _max in cerebral cortex, plasma and liver of experimental rats when compared with control without having significant change in brain stem but with decreased K _m (P < 0.001). Our results suggest that decreased function of dopamine mediated through DA D₂ receptor in the cerebral cortex and brain stem enhanced the brain, plasma and liver ALDH activity in ethanol treated rats. This ALDH regulation has significance to correct alcoholics from addiction due to allergic reaction observed in aldehyde accumulation.     

Cold resistance of the brain during hibernation: I. K+ transport in cerebral cortex slices

The brains of the hibernating hamsters and 13-lined ground squirrels maintain Na⁺ and K⁺ at the same concentrations as in the awake state. The ability of slices of the cerebral cortex when incubated in vitro to accumulate or retain K⁺ is similar in the awake hamster and rat at both 38 and 5 ° C. On the other hand, slices of cerebral cortex from the hibernating hamster retained slightly more K⁺ at 5 °C than did those of awake hamster or rat. It was concluded that the cerebral cortex of the awake hamster is probably not cold resistant with respect to the maintenance of cation balance. Further, the cold resistance that exists in the cerebral cortex of the hibernating hamster is largely destroyed when the brain is disrupted by slicing.