Chemical-Induced Brain Injury

Mohamed B. Abou-Donia, Ph.D.Department of Pharmacology and Cancer Biology

Duke University Medical CenterDurham, North Carolina 27710

donia@duke.edu

Brain Injury

1. Little is known about the etiology of many brain diseases, such as Alzheimer's Disease, Parkinson's Disease, Multiple Sclerosis, Amyotrophic Lateral Sclerosis (ALS), or Autism.

1. Chemicals can cause brain injuries that resemble brain diseases, for example:

a. Manganese causes brain injury similar to that of Parkinson’ disease

b. Organophosphate-Induced Delayed Neurotoxicity (OPIDN) has been misdiagnosed as Multiple Sclerosis.

The Brain

The brain contains approximately

200 billion cells (neurons) and

a trillion supporting cells.

Brain neurons are all formed before

birth, and no new neuronal cells are

born after birth.

The Brain

The brain requires one fifth of the oxygen and

energy (glucose) consumed by the body to

maintain its function.

The hourly flow of blood through the brain is

approximately 13 gallons, which accounts for

one fifth of the blood pumped by the heart.

Brain Regions

The brain is divided into three sections:

a. Forebrain: cerebrum, limbic system (amygdala,hippocampus, septum), thalamus, hypothalamus.

b. Midbrain

c.Hindbrain: Pons, medulla oblongata, cerebellum

Cerebral Cortex

• Language

• Vision

• Higher-order processing

• Movement

Cerebral Cortex II

• Corpus striatum• Degenerates in

Parkinson’s– Paralysis– Loss of sensory

input– Loss of reasoning,

judgment, memory, etc

Limbic System

• Emotion

• Memory– Consolidation– Storage– Working

memory

• Movement

Limbic System

• Emotion

• Memory– Consolidation– Storage– Working

memory

• Movement

Cerebellum

• Motor learning

• Posture

• Gait

Brain Supporting Cells

Supporting cells continue to divide throughout life.A. Glial Cells:1. Astocytes or astroglia: provide structural

(Blood Brain Barrier) and nutritive support and form “glial” scar after injury.

2. Oligodendrocytes or oligodedroglia: form myelin sheath of axons in the CNS.

3. Microglia: Activated after injury and act as scavengers taking up debris.

B. Endothelial Cells:Form the walls of brain capillaries and the BBB.

Blood brain barrier

Blood Vessel

Blood brain barrier

General capillary Brain capillary

Fenestra

Intercellular clef

Endothelial cell

Pinocytotic

vesicles

Mitochondria Endothelial cell

Astroglial

process

Pericyte

Basement

membrane

Tight

junction

The Blood Brain Barrier (BBB)

Is formed by brain capillary walls endothelial cells:

1. Tight junctions

2. No fenestras

3. Few pinocytotic vesicles in the cytoplasm (BChE)

4. Increased mitochondria (active transport)

5. Basement membrane (AChE)

6. Astrocytic feet ensheathe 95% of endothelial cells

7.Pericytes with smooth muscle-like properties

8. P-glycoprotein (P-gp) to remove undesired substances.

Nervous System-Specific Proteins

1. Axons a. Neurofilament proteins (NFP)

b. Tau proteinsc. Tubulin

2. Dendrites Microtubule Associated Proteins-2 (MAP-2)

3. Myelin Myelin basic protein (MBP)

4. Astrocytesa. Glial Fibrillary Acidic Proteins (GFAP)b. S-100 protein

The Cytoskeleton

1. The cytoskeleton in the neuron consists of straight and parallel:a. 25-nm microtubules (α- and β-tublin)b. 10-nm neurofilaments c. Microtubule associated proteins (MAP-2 and Tau) that function as cross-bridges to link microtubules and neurofilaments.

2. The cytoskeleton gives the cell its shape and transport material within and outside the cell.

Neurofilament Proteins

Neurofilaments consist of 3 polypeptides:1. A 200-k Da, outer or high molecular weight

protein (NFH),2. A 160-k Da, middle or medium molecular

weight protein (NFM), and3. A 70-k Da, core and low-molecular weight

protein (NFL).NFH is peripherally located, and especially

vulnerable to injury and is an early marker to neuronal damage.

Tau Proteins

Tau Proteins are:

• Present almost exclusively in the axon.

• Involved in microtubule assembly and stabilization.

Tubulin

1. Tubulin is present in all cells

2. It is present at high level in the brain where it comprise approximately 10% of brain protein.

3. Testes have high contents of tubulin

MAP-2

Microtubule Associated Proteins-2 (MAP-2):

1. Are found exclusively in the somato-dendrites of the neurons

2. They promote polymerization and stabilization of microtubules.

Myelin Basic Protein (MBP)

MBP is a major constituent of the myelin that is formed by:

1.The supporting cells, oligodendrocytes in the central nervous system and

2. Schwann cells in the peripheral system.

Astrocytic Proteins

Astrocytes form the following proteins:1. Glial Fibrillary Acidic Protein (GFAP) is

secreted following axonal injury to form gliotic scar.

2. S-100 is a calcium binding proteins that is formed in response to acute brain injury such as brain infarction and has been used to assess ischemic brain damage.

Autoantibodies against Brain Specific Proteins

1. Normally, small amounts of brain-specific proteins may leak into the circulation, where they react with B lymphocytes to form autoantibodies that are reactive against these proreins.

2. Autoantibodies are increased with age.3. Damage to neuronal and glial cells in the brain

or of the blood brain barrier (BBB), causes more leakage of these proteins into blood stream, with subsequent increased formation of the autoantibodies against them.

Brain-Specific Protein Autoantibodies: Biomarkers for Neurological Diseases

Brain-Protein protein autoantibodies havebeen detected in the sera of patients with:1. Alzheimer’s Disease2. Parkinson’s Disease3. Myasthenia Gravis4. Multiple Sclerosis5. Kuru Disease6. Creutzfeldt-Jacob Disease, and 7. Picks Disease8. Down Syndrome

Chemical-Induced Axonal Degeneration and Gliosis in Animals

Axonal and gliosis are induced in animals by:

1. Organophosphates (Abou-Donia, 1982)

2. n-Hexane, MBK (Lapadula et al, 1988)

3. Carbon disulfide (Wilmarth et al., 1993)

4. Acrylamide (Reagan et al., 1994)

5. Glycidamide (Reagan et al., 1995)

Mechanisms of Neurotoxicity

• Nonspecific: lack of oxygen (hypoxia)

• Selective: Chemicals can target:– Nucleus– Axon– Myelin– Synapse

Hypoxia

• Anoxic– Respiratory paralysis– Failure of blood to carry oxygen

hemoglobin• Ischemic

– Cardiac arrest– Hypotension (vasodilation)– Hemorrhage/thrombosis– Carbon monoxide

• Cytotoxic– Cytochrome oxidase inhibition– Metabolic inhibition– Repeated hypoxia (e.g., TIA)

Selective neurotoxins

• Cell body• Synaptic• Axon

– Central-peripheral proximal axonopathy

– Central-peripheral distal axonopathy

• Neurofilamentous• Tubulovesicular

– Conduction

Chemical-Induced Neurodegeneration

The following chemicals caused neuronal degeneration in the cerebral cortex, hippocampus, and cerebellum of brain of exposed rats.

1. Organophosphorus Compounds:Sarin, Malathion, Chlorpyrifos, TOCP, TmCP, TpCP.

2. Pyrethroids: Permetrhrin

Increased Autoantibodies Against Brain-Specific Proteins

1. Axonal DegenerationNeurofilament proteins, Tau, tubulin

2. Demyelination Myelin Basic Protein (MBP)

3. Dendrite DegenerationMAP-2

4. AstrogliosisGFAP

5. Acute Brain injuryS-100

Consequences of Axonal Degeneration

Increased autoantibodies against neurofilaments, tau,tubulin or/and MBP indicate axonal degeneration.

Degeneration in the cerebral cortex leads to:

1. Motor and sensory abnormalities, 2. Ataxia,3. Deficit in posture, locomotion, and skilled movements 4. Fine motor movements (fingers, speech, facial

expression, etc., 5. Weakness

Consequences of Axonal Degeneration

Axonal degeneration of the limbic system

including the hippocampus leads to:

1. Learning and memory deficits

2. Neurobehavioral (mood, emotion and judgment) abnormalities

Consequences of Axonal Degeneration

Increased autoantibodies against MAP-2

suggests damage to the dendrite-rich

Purkinje cells in the cerebellum resulting in:

1. Gait and coordination abnormalities

2. Staggering gate

3. Ataxia

Consequences of Gliosis

Increased autoantibodies against GFAP suggests:

1. Axonal injury (forms scar).

2. Neuropsychiatric disorder.

Consequences of Gliosis

Increased autoantibodies against S-100 suggest:

1. Traumatic brain damage2. Acute phases of brain injury such as brain

infarction, and3. It as been used to assess ischemic brain

damage. 4. Can help to differentiate between acute

and chronic brain injury.

Hypothesis: Increased Autoantibodies Against Brain Specific Protein

Hypothesis:1. Following neuronal injury, neuron- and glia-

specific proteins are released into circulation.2. Released necrotic neuronal and glial elements

accumulate and stimulate B lymphocytes to produce autoantibodies that are reactive against these proteins.

3. Increased autoantibodies against brain-specific proteins in the serum are indicative ofneuronal damage.

Significance

Antibody test that could detect ongoing or at-risk status of neurodegenerative diseases would be desirable, because:

1. Antibodies are extremely sensitive and specific measure

2. They can amplify the signal of an altered biological environment.

Specific Aim

To correlate neurological deficits in persons following chemical exposure with sera levels of autoantibodies against brain neuronal and glial specific proteins.

Methods1. Patients. Individuals exposed to pesticides, industrial chemicals, and flight cabin

fumes. 2. Sera from the patients and healthy controls were obtained.

3. Western Blotting. Standard brain-specific proteins are separated on SDS –PAGE. 4. Proteins were transferred into PVDF membranes.

5. Membranes were incubated with serum samples at 1:50 dilution. 6. After washings, the membranes were incubated with horseradish peroxidase-

conjugated goat anti-human IgG.

7. The membranes were developed by enhanced chemiluminescence.

8. The signal intensity was quantified.

9. The results were normalized to sera albumin.

Results

1. We have confirmed the strong association between levels of autoantibodies against brain-specific proteins and chemical-induced neurological deficits.

2. Increased autoantibodies were more frequent among the patients than the controls.

3. The results indicate axonal and dendrite degeneration followed by demyelination of brain neurons.

Conclusions

In the absence of neurological diseases, while not diagnostic for a specific illness, the presence of increased circulating autoantibodies against neuronal and glial proteins is consistent with, and can be used as further confirmation for chemical-induced injury.