(A review article by Dr. RAJ KUMAR DHAUGODA FROM NEPAL-
MBBS, MD IN FAMILY MEDICINE AND DIALECTICAL MATERIALIST)
Clinical features of Hypoxic-Ischemic Encephalopathy
· Neonatal seizure - over stimulation of excited neurons and reciprocal membrane depolarization
· Resp. depression, poor suck - impaired action of neurotransmitters in brain stem.
· Coma - Metabolic disrupt of reticular activating system
· Brain edema - Disturbed - ability to pump water at of hugely- breakdown of blood brain barriers.
Patho-physiology of Hypoxic- Ischemia Brain Injury and chemical changes
Main 3 mechanism –
-Hypoxic- hypoxic (decreased O2 concentration)
-Anemic hypoxia (decreased O2 delivery)
-Ischemia hypoxia (decreased Blood flow)
· Global Ischemia - results from inadequate blood flow.
E.g. during cardiac arrest.
Or sever hypoxia, severe hypotension.
· Focal Ischemia - Results from local disruption of Blood supply e.g. ischemia / stroke, vascular anomalies.
1.Cerebral blood flow :-
Reduction of CBF 15ml / min / 100 gram (CBF) results in failure of electrical activity severity of the hypoxic - Ischemic insult is a function of both the duration and degree of CBF reduction. Delayed Hypo perfusion (which is occurred in post ischemia) is the result of brain injury rather then the cause.
2)Histological changes: -
After prolonged global ischemia, there are seen mitochondria, endoplasmic reticulum, per neuronal & astrocytic swelling. Also develops eosinophilia & intracellular organelles becomes disrupted. The disruption of organelles is evidenced by the loss of Nissl substances, the formation of ribosomal rosettes, and swelling are reversible, DNH fragmentation is a characteristic of apoptosis, has been demonstrated after both focal and global Ischemic insult, found in CA1 region of hippocampus.
During Hypoxic- Ischemic Brain injury.
(1)Adenosine 5 - Triphosphate Depletion (ATP).
· ATP is needed for numerous cell metabolism, occur very early stage.
· Most importantly needs for preservation of cell membrane ionic gradients.
· The cause of ATP depletion is the - After hypoxia or ischemia. Oxygen is depleted and oxidative phosphorylation stops ATP depletion leads to membrane depolarization and massive Tran membrane ionic fluxes. Lock of Na+ / K+ ATPase activity leads to net influx of Na+, chloride, and calcium and net efflux of potassium.It is reversible if the Ischemic insult is of limited duration.
· During complete global ischemia, a substrate delivery to the brain is interrupted. The oxygen supply is depleted almost immediately. But glucose is available to the brain for a short time during the ischemic period via the breakdown of glycogen. Since glycogen storage in brain is small and anaerobic degradation of glucose is relatively inefficient, the energy obtained by this metabolism is not sufficient for normal brain metabolism. As glycol sis produces lactate and H+, causes tissue injuries (due to acidosis). Hence it is very danger to give glucose during ischemic & Hypoxic brain injury - more probes to produce lactate causing more acidosis. Peri ischemic hyperglycemia has been shown to result in a more pronoun ad fall in both intracellular and extra cellular brain PH during ischemia.
· The mechanism of acidosis- Induced injury- Direct cytotoxicity, cellular swelling, oxygen- radical mediated damage, and alterations in intracellular calcium homeostasis.
· Acidosis may promote cellular swelling by inhibition ATP production and there by reduce Na+ /K+ ATPase activity.
· Acidosis also promotes the change of super oxide radical (O2) to more lipid soluble form (HO2*). Acidosis also promotes nitric oxide (NO) coupled production of hydroxyl radicals to form peroxynitrite.
(3)Excitatory Amino acid (NMDA receptors), (non- NMDA receptor) (AMPA )
· NMDA (N-methyl- D- aspartate) receptor is an ionotropic (Excitatory) glutamate receptor. Glutamate is an excitatory neurotransmitter, as mediator of hypoxic- ischemic brain injury. Extra cellular glutamate level in bran is increased during ischemia, dramatically and after 30 minute of ischemia, exceeds by six fold the concentration required to kill neurons after 5-minute exposure in vitro. Glutamate receptor antagonists have been shown to ameliorate injury in animal models of focal and global ischemia, Finally glutamate receptors antagonists attenuate injury of cultured neurons when the neurons are exposed to oxygen deprivation.
· The NMDA receptor is the most widely studied glutamate receptor. The channel gated by this receptor allows entry of sodium, potassium and calcium, and calcium accumulation via this channel is felt to be responsible for the neuro toxicity of glutamate.
· The NMDA receptor- ion channel complex is unique in that calcium entry modulated by several factors including. Glycine, polyamines, magnesium and zinc
· The non- NMDA receptor (AMPA/kainate): - alpha- amino- 3- hydroxy- methyl- 4- Isoxazole- 4- pripionic acid. This channel is permeable to only monovalent cations (Na+ / K+) and relatively impermeable to calcium ion.
But in ischemic injury, the structure of this non-NMDA receptor is altered and influx of Ca++ is occurred causing more cell damage.
· Metabotropic Glutamate receptors: - are coupled to Adenyl cyclase or phospholipase- C via G-proteins. G-proteins are membrane proteins that facilitate Trans- membrane signaling. Thus, stimulation of the Metabotropic receptor leads to increased production of diacylglyceral and inositol triphosphate (IP3) or decreased production of cyclic adenosine monophosphate (cAMP) seen subtypes of the Metabotropic glutamate receptor have been identified e.g.
· M GluR1 stimulates phospholipase-c
· M GluR2 inhibits adenylate cyclase
· M GluR3 inhibits adenylate cyclase
· M GluR4 inhibits adenylate cyclase
· M GluR5 Stimulates Phospholipase - C
· M GluR6 Inhibits adenylate cyclase
· M GluR7 Inhibits adenylate cyclase
Calcium plays a pivotal role in glutamate neurotoxicity, proposed that glutamate - induced neuronal death is a three-stage process e.g.
b) Amplification and
During inductions, glutamate activates a neuronal receptor activation of NMDA receptor leads to an influx of Calcium, where as activation of non-NMDA receptors leads to influx of sodium and obligatory influx of chloride and water, and also influx of Ca++. Stimulation of the Metabotropic receptors leads to increased production of IP3 and Diacylglycerol. The cellular injury cascades initiated during the induction stage can be divided into two components .
a) Acute cellular swelling (sodium dependent) and
b) Delayed neuronal degeneration (calcium dependent)
Amplification Results - from the damaging cascades initiated by increased concentrations of Ca++, IP3 and Diacylglycerol. Altered calcium homeostasis leads to activation of phospholipase, proteases, endonucleases, protein kinese and calmodulin - regulated enzymes e.g. Nitric oxide is an important mediator of glutamate neurotoxicity. IP3 causes release of sequestered intracellular calcium, resulting in further increase in intracellular Ca++ concentrations, and Diacylglycerol causes activation of protein kinase - C.
The expression stage - consists of actual cell destruction resulting from activations of catabolic enzyme system and oxygen radical production.
(4)Calcium metabolism: -
· Role in the regulation of many cellular metabolic processes, and there fore, normally, the concentration of cytosol free calcium is tightly controlled.
· In hypoxic - ischemic injury of brain, there is impaired intracellular calcium homeostasis, resulting in massive increases in the intracellular calcium concentration.
· This intracellular calcium accumulation causes irreversible cellular injury by activating phospholipase, proteases, and endonucleases and uncoupling oxidative phosphorylation.
· Recent works suggests that calcium play a key role in triggering nitric oxide syntheses, protein kineses, oxygen radical production and alteration in gene expression.
· It has been exclusively demonstrated that intracellular calcium accumulation occurs during and after Ischemia. Evidence for role of calcium in the pathophysiology of ischemic injury exists in the form of numerous studies that demonstrate that pharmacological blockade of calcium entry during and after global ischemia confers protection (improves outcome).
· Several vitro studies provide more direct evidence favoring a role for calcium accumulation in ischemic cell death. Removing calcium from the extra cellular space has been shown to decrease cellular injury in neuronal and astroglial cultures exposed to oxygen and glucose deprivation.
· There are two major types of calcium channels located in the cell membrane of neurons:-
Receptor operated calcium channels e.g.
1. NMDA – receptor
2. Non-NMDA – receptor
3. Voltage sensitive calcium channels e.g. L, N, P & T type
Long lasting (L-type) - High voltage (activation) - function is dihydropyridine sensitive. N-type - (Function - neuro transmitter release, P-type (function - neuro transmitter release), & T-type (transient) - low voltage type, rapid inactivation occurred function is as pacemaker activity.
Cerebral ischemia is accompanied by a significant decrease in extra cellular calcium concentration, which is consistent with an intracellular shift of calcium.
· Energy failure (depletion of ATP) during cerebral ischemia results in membrane depolarization, which allows calcium influx via voltage sensitive calcium channels also and causes glutamate release. Glutamate can promote calcium influx by three different mechanisms namely
· Via opening NMDA receptors gated calcium channel
· Via stimulation of Non-NMDA receptors opens Na++ channels and resulting in massive influx of Na++ and subsequent membrane depolarization. These membrane depolarizations then allow calcium entry via voltage - sensitive Ca++ channels.
· Via the ion channel gated by Non-NMDA receptors allows direct influx of calcium.
In summary, intracellular calcium accumulation is assumed to play a significant role in ischemic brain injury, and its accumulation appears to be initiated by energy failure (ATP depletion) and glutamate - mediated calcium influx.
(5) Phospholipid hydrolysis: -
· Metabolism of membrane phospholipids is to play a key role in the pathophysiology of ischemic brain injury.
Early in the cause of ischemia, free fatty acids are released from membrane phospholipids by enzymatic hydrolysis.
Fatty acids appears to be released via two pathways –
· Membrane phospholipid degradation by the action of phospholipase A1 and phospholipase A2
· Degradation of phosphoinositides by the action phospholipase C, leading to production of diaglycerol, which is then, hydrolyzed by Diacylglycerol and monoacylglycerol lipases
· Energy depletion (ATP depletion) leading to a greater rate of deacylation relative to reacylations (energy dependent) is one during ischemia. Stimulation of NMDA receptors leads to activation of phospholipase A2, this activation appears to be calcium mediated.
· Farooqui and Colleagues - have shown that NMDA receptors stimulation also leads to activation of monoacylglycerol and diacylglycerol lipases.
· Diacylglycerol is a substrate for further free fatty acid release and by activating protein kinase -C. IP3, mobilizes intracellular calcium and destructed to neuron cells and diacylglycerol activate protein kinas C and inhibit Na+ / K+ ATPase and promote brain edema.
In addition, release of free fatty acids at the onset ischemia provides the substrate (arachidonic acid) for production of eicosanoids and oxygen radicals during reperfusion.
(6) Eicosanoids Metabolism: -
· A arachidonic acid released at the onset of ischemia becomes the substrate for eicosanoids production during reperfusion.
· Products of cyclooxygenase pathway i.e. thromboxane and prostaglandin and the lipoxygenase pathway i.e. leukotriens are formed during reperfusion after ischemia. Thromboxane causes vasoconstriction and platelet aggravation and causes delayed hypo perfusion. Leukotriens also causes vasoconstriction of cerebral vessels.
· In addition, the cyclooxygenase and lipoxygenase pathways produce super oxide radicals (oxygen radicals during reperfusion).
(7)Oxygen radicals: -
· Oxygen radicals have been implicated in numerous disease processes, including pulmonary oxygen toxicity, carcinogenesis and perfusion injury after ischemia.
· Indirect evidence in support of oxygen radical mediated injury after cerebral ischemia includes decreases in tissue antioxidant levels during ischemia and reperfusion, increased productions of lipid peroxides during reperfusion, improved outcome after Global and focal ischemia in animals treated with antioxidants, and reduction in infarct volume.
· Radicals are molecules that contain at least one unpaired electron in the outer (valance) electron orbital. Some of the more common oxygen radicals include super oxide radical (O2.-), hydroxyl radical (OH.), and hydrogen peroxide (H2O2.). These molecules are very reactive and as a result very short-lived.
· Potential mechanisms of oxygen radical production during reperfusion injury include oxidations of hypoxanthine by xanthine oxidase, and lipoxygenase pathways.
· ATP is metabolized through a series of intermediates to hypoxanthine, the substrate for xanthene oxydase. Adenosine and hypoxanthine concentrations in brain increase dramatically within seconds after the onset of Ischemia.
· More importantly, ischemia has been shown to induce the conversion of xanthine dehydrogenisis to xanthine oxidase, leading to significant levels of xanthine oxidase in brain. This conversion appears to be a calcium dependent process, convey vascular endothelial damage. Recent work by Phillis and Sen provides direct evidence that the xanthine oxidase pathway contributes to oxygen radical production during perfusion. They demonstrated that oxypurinol, a xanthine oxydase inhibitor, significantly decreased production of hydroxyl radical after cerebral ischemia in rats.
· Lipid peroxidation - is an iron-dependent process in which oxygen radicals oxidative rearrange the structure of double bonds of unsaturated fatty acids of membrane phospholipids. A hydrogen atom is removed from polyunsaturated fatty acids; thus can only do by the hydroxyl radical, Alternately, this lipid peroxidation process from malandialdehyde (a lipid peroxide radical). These lipid peroxidation reactions are intensified in the presence of calcium and acidosis. Lipid peroxidation C and inhibit Na+ / k+ ATPase. Causing cellular injury during reperfusion, after cerebral ischemia.
(8)Nitric Oxide: -
· The function of nitric oxide in CNS-It is appeared to be involved in regulations of cerebral blood flow and in cell-to-cell signaling. In some neurons, nitric oxide synthesis activity is regulated by the NMDA-receptor. NMDA receptor stimulation results in calcium influx and activation of nitric oxide syntheses. Nitric oxide is produced and then diffuses to target cells and stimulates guanidine cyclase leading the production of cyclic GMP. GMP then produces the physiological effect, e.g. Vase relaxation cell signaling.
· Nitric oxide stimulates macrophage, microghia and astrocytes in response to stimulation by cytokines, causing cytotoxicity.
· Nitric oxide appears to be a mediator of glutamate neurotoxicity. It is postulated that NMDA receptor over stimulation leads to massive calcium influx and activation of nitric oxide syntheses. Nitric oxide produced via this mechanism contributes to the cytotoxicity of glutamate.
· Nitric oxide is produced during both focal and global cerebral ischemia. It is detrimental to reasons if produces more hydroxyl radicals.
(9) Protein phosphorylation: -
· Many cellular processes are regulated by protein phosphorylation.
· The Phosphorylation State depends on the relative activities of two opposing types of enzymes - kinase and phosphates.
· Kinesis add phosphate groups to protein where as phosphates remove phosphate groups from protein.
· Second messengers such as calcium, diacylglycerol, and acid activate protein kinese.
· Protein kinase regulates important neuronal functions, including ion channel activity, transmitter release, receptor function, gene expression and protein synthesis.
· These important kinases have been shown to have changes in activity after cerebral ischemia.
a) Protein kinase - C,
b) Calcium - calmodulin dependent kinase - II
c) Casein kinase -II
· Activation of protein kinase - C requires translocation from cytosol to cell membrane; this translocation is promoted by a rise in the intracellular concentration of calcium. After association with membrane phospholipids, protein kinase - C becomes fully activated in the presence of diacylglycerol.Calcium - calmodulin dependent kinase - II acting is decreased after ischemia, during ischemia its activity is increased.
(10)Protein synthesis: -
· Protein Synthesis is essential for cell survival. Proteins are involved in nearly every process in the cell e.g., Enzymatic reactions, transport, storage, intracellular sygnalling, intracellular signaling and cytoskeletal support.
· Hence, protein synthesis is very important for cellular recovery after hypoxic ischemic injury.Kleihues and Hoss Mann 1st demonstrated that total protein synthesis is impaired after cerebral ischemia and reperfusion.
· Protein synthesis requires: -
Energy (ATP, GTP)
Functional transcription mechanism
Processing and transport of m RNA from the site of transcription to site of translation
Functional translation mechanism
· As the concentration of ATP, GTP is rapidly decreased during ischemia, and this is adequate to explain inhibition of protein synthesis during ischemia and reperfusion.
· Recovering of protein synthesis is correlated with cell survival.
(11)Gene expression: -
There is increased expression of C-for and C-jun has been demonstrated after global cerebral ischemia.
Increased synthesis of heat in the protective action.This HSP70 protein in the protective action
(11) Inflammation: -
Dietrich et al.were able to demonstrate polymorph nuclear leukocyte infiltration; in rat model of global ischemia. And increased level of TNF-alpha.
POTENTIAL THERAPIES( current practices of Brain resuscitation)
1. Barbiturates: -
· Decrease seizure
· Decrease intracranial pressure
· There is some evidence of giving thiopentone 120 mg / kg IV after 30 & 60 minutes of post ischemia, outcome is ameliorates of brain damage, After global ischemia in monkeys.
· But same type of study did not show the thiopental amelioration of brain damage.
· in summary. Barbiturates have been proposed for use after global ischemia. But it is not supposed by data obtained in animal and human.
2. Super oxide dismustase: -
· Dimutation reaction of super oxide radical, The capper zinc super oxide dismustase has been proposed as therapeutic agent for reperfusion injury, because of its ability to scavenge oxygen radicals.
· Recently, a novel supervise dismustase derivative, polybutyl ester covalently linked to super oxide dismustase, has been shown to ameliorate delays hypo perfusion and improve neurological out come after global ischemia in dogs.
· 21-amino-steroids (oxygen radical scavenger) a portent inhibitor of lipid peroxidation but lack classical steroidal activities
· to summary: it is obvious that there is no current role for oxygen radical scavenger therapy in children with brain injury. There is only preliminary evidence supporting the efficacy of this agent after a global ischemic insult.
3. Calcium channel Antagonist:
· The use of Calcium Channel blockers after global cerebral ischemia is considered e.g. flunarizine, lidoflazine, and nimodipine.
· Nimodipine shows increase survival rate up to 12 months after global cerebral ischemia.
4. Glutamate Antagonists:
E.g. dizocilpine maleate _(NMDA receptor antagonist)
NBQX (2,3 dihydroxy –6-nitro-7-sulfamoxylbenzoquinoxaline)
(Non- NMDA receptor antagonist)
5.Nitric oxide syntheses inhibitors:
No satisfactory out came has been reported with N-omega -nitro –L-arginine.
In the global ischemia, the temperature out came is that hyperthermia. (During intra and post ischemic period)
In study it is shown that intra ischemic hypothermia is protective in both focal and global cerebral ischemia
Post ischemia hypothermia is protective after global ischemia provided that it is initiated very soon after the inset of reperfusion
(Ref: Mark c. Rogers, David G Nichols, Text book of Pediatric intensive Care, 3rd edition, 1999, chapter 20, theories of Brain Resuscitation, by Steven E. Haun, Jeffrey R. Kirsch & Michael Dean, page 699-733).
1. The approach to the patient with global Hypoxic Ischemic brain injury is a difficult task, by using above drugs separately gives the incomplete results and not fully satisfactory result.
2. It is necessary to understand the more detailed patho- physiology of neuronal damage and recovery & its limitations, information of time, space,motion and stress factor magnitude of ischemic damage.
3. It is necessary to ongoing research & correlation of newer knowledge for newer more effective drugs( magnesium compound) or techniques e.g. gene therapy,stem cell therapy etc.