Two different coma models

Yaohui TangMax-Planck-Institute for Biological Cybernetics

What is coma?• Coma is a state of unconsciousness, which is marked by a lack

of awareness and response to external stimulus.

Steven Laureys, et al, The Lancet, 2004

Cerebral metabolism in different brain states

Steven Laureys, et al, The Lancet, 2004

Conscious

Locked-in syndrome

MCS

Vegetative state

Important pathways and brain nucleus for arousal

• Reticulo-thalamo-cortical pathway (widely accepted)

Basilar artery occlusion (BAO)

• Thalamus-Basal forebrain (BF)-Parabrachial nucleus-precoeruleus area (PB-PC)

Neurotoxin stereotaxic injection (NSI)

Basilar artery

• One of three major arteries feeding

the circle of Willis

• Situated on the ventral surface of the

brainstem

• Supplies the major portion of the blood

flow to the brainstem

SCA: superior cerebellar artery

AICA: anterior inferior cerebellar artery

PICA: posterior inferior cerebellar artery

Tracey Baird, et al, Neurocritical Care, 2004

Basilar artery occlusion

Single point occlusion

Two points occlusionD-M

M-P

D-P

Purpose: develop a reproducible rat model of brain stem ischemia

• In 11 rats, the basilar artery was occluded at a single point along its length.

• 12 rats underwent occlusion at twopoints 3 mm apart at various sites along the length (above AICA or below AICA)Basilar

artery

ResultsSingle-point or two-point BAO reduced peak-to-peak amplitude of the cortical SEPs by >50% within 15 minutes

The SEPs gradually recovered over 3-4hours, and the response amplitudes exceeded baseline values in seven of the 17 rats by 4 hours after occlusion.

By 24 hours after basilar artery occlusion,amplitudes and latencies returned to baseline values.

HE staining

Two-point BAO below AICA Two-point BAO above AICA

No infarct in any rat with single-point basilar artery occlusion

Two-point occlusion above or belowthe AICA produced brain stem infarcts

Conclusion

• Basilar artery occlusion at any single point between the foramen

magnum and the circle of Willis in 11 rats did not produce

histologically detectable infarcts in the brain at 12-24 hours.

• Two-point occlusions of the basilar artery in 12 rats produced

variable infarcts between the occlusion sites but no ischemic lesions

elsewhere.

• Basilar artery occlusions invariably suppressed cortical somatosensory evoked potentials by >50%.

Background

• Arousal pathway passed through the paramedian midbrain reticular formation and bifurcated at the diencephalon into two branches, into the thalamus and hypothalamus

• Most neurons participating in these pathways from the rostral pons and caudal midbrain:

Noradrenergic locus coeruleus

Serotoninergic dorsal pedunculopontine

Laterodorsal tegmental nuclei

Parabrachial nucleus

However their functions in awake/sleep are unknown.

Methods

1st part

• Pathogen-free adult male Sprague-Dawley rats (275–300 g)

• Lesions of the thalamus: injecting 50 nl of a 10% solution of ibotenic acid bilaterally

• Lesions of the basal forebrain: injecting a 0.1% solution of either IgG192-saporin or orexin-saporin (OX-SAP) at four different sites (ibotenic acid cause rats to die; high dose (125ng) OX-SAP kill all noncholinergic neurons and 88% of cholinergic neurons; 100ng OX-SAP kill all noncholinergic neurons and 19% of cholinergic neurons)

• To kill cholinergic BF neurons specifically, 1ug IgG 192-saporin was injected into the lateral ventricle.

• EEG/EMG were continuous recorded on day 7 postoperatively.

• c-Fos immunohistochemistry (an indirect indicator of neurons firing, it gives a rough indication of the degree to which neurons have been receiving excitatory inputs that elevate cyclic AMP or intracellular calcium)

2nd part

• Using cholera toxin subunit B (CTB) to retrogradely trace inputs to the BF and thalamus from sites in the brainstem to define the cell groups

• In situ hybridization for the vesicular glutamate 2 transporter (VGLUT2), to determine which of these cells were likely to be glutamatergic.

3rd part

• Using local injections of orexin-saporin to ablate neurons in the parabrachial nucleus and precoeruleus region

• EEG/EMG were continuous recorded at 7 days postoperatively.

• c-Fos immunoreactivity

ResultsIbotenic acid induced lesions of the thalamus

Effects of thalamus lesions on c-Fos expression and Sleep-wake behavior

A slight decrease in theta power during the subjective night after thalamus lesion

Summary 1

• Thalamus lesions, even with an extensive lesion, did not affect EEG/EMG pattern, sleep/wake pattern, c-fos expression and behavior, except a slight decrease in theta power during the subjective night.

• No coma-like syndrome was observed.

Nonselective lesions of the BF

10 days after OX-SAP, 7/11 rats exhibited a coma-like state• EEG at all times was dominated by sub-delta (<1 HZ EEG) activity.

Effects of BF lesions on the c-Fos expression

A dozen cholinergic cells are the only surviving Neurons

Minimal c-fos expression in neocortex High expression in TMN and LC

C-Fos expression on nonselective and selective lesions of the BF

By continuous gentle touch, rats maintained a tonically active EMG.EEG showed a monotonous slow-wave activity

Effects of BF lesions on the EEG pattern induced by continuous stimulation

No coma-like behaviors were induced in Ch BF lesions or Non-Ch BF lesions

Summary 2

1. BF is a critical relay for maintaining the waking pattern of behavior, EEG and cFos expression.

2. Both cholinergic and noncholinergic (mainly GABAergic) BF neurons work jointly in control of cortical arousal. Either component alone is capable of supporting cortical arousal.

Source of inputs to the BF neurons?

Retrograde tracer CTB injection to S1 to search which provides arousal inputs to the BF

• Large numbers of neurons in the medial PB (MPB)

• Small number in the PC

• Almost all the CTB labeled cells in the PC and PB also expressed VGLUT2, indicating PB/PC provide glutamatergic inputs to the BF

PB/PC lesions induced by OX-SAP injection

Comatose happened 10 days after injection

LPB and MPB lesions increased sleep

Bulk of EEG power was <1 HZ after PB/PC lesion

Low level of c-Fos in the neocortex

Effects of PB/PC lesions on the EEG pattern induced by continuous stimulation

By continuous gentle touch, rats maintained a tonically active EMG. EEG showed a monotonous slow-wave activity

Summary 3

1. PB/PC is critical for achieving and maintaining an activated EEG and a waking state.

2. PB/PC-BF-neocortical axis controls neocortical arousal

Important points of the study

• 1. Challenge widely accepted view of comatose model (thalamus)

• 2. Provide solid evidence that PB/PC-BF-Cortex may constitute a critical pathway for maintaining a waking cortical state.

Thanks for your attention