Two different coma models Yaohui Tang Max-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. Brain death: the loss of all brainstem reflexes coma is a state of unresponsiveness in which the patient lies with the eyes closed, cannot be aroused, and has no awareness of self and surroundings. Patients in a vegetative state are awake but are unaware of themselves or their environment MCS: patients who are not in a vegetative state but are unable to communicate consistently. Locked-in syndrome is defined by sustained eye opening. awareness of the environment, aphonia or hypophonia, quadriplegia or quadriparesis, and vertical or lateral eye movement or blinking of the upper eyelid to signal yes/no responses. Eye or eyelid movements are the main method of communication. Steven Laureys, et al, The Lancet, 2004

Cerebral metabolism in different brain states Conscious Locked-in syndrome MCS Vegetative state Normal people has the strongest cerebral metabolism, while VS has almost no cerebral metabolism Steven Laureys, et al, The Lancet, 2004

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 Basilar artery is one of three major arteries feeding the circle of Willis, which is situated on the ventral surface of the brainstem and supplies the major portion of the blood flow to the brainstem. It is joined to the brainstem via penetrating median, paramedian, and short and long circumferential branches, supplies blood to many large and small vessels in the posterior circulation, which is very important for consciousness Tracey Baird, et al, Neurocritical Care, 2004

Basilar artery occlusion Single point occlusion Two points occlusion D-M M-P D-P Distal: above SCA Mid: AICA-SCA, BAO Proximal: Below AICA

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 two points 3 mm apart at various sites along the length (above AICA or below AICA) Basilar artery A 3-cm midline neck incision was made, and the muscles were divided in the midline to expose the trachea. A polyethylene tracheostomy tube (PE240) was inserted in the first 17 rats and sutured in place. The last 12 rats were orotracheally intubated with a polyethylene tube (PE160) under direct visualization after the midline incision. After dividing the right sternohyoid muscle, the carotid bundle was exposed, separated from the paratracheal structures, and retracted laterally, using an approach described in larger animals.6 The anterior cervical muscles were then divided in the midline and detached from the skull base. This exposed the skull base from the foramen magnum to the posterior occipitosphenoid suture rostrally and to the occipital condyles laterally. A high-speed drill was used to thin the exposed basilar bone. The inner table was carefully elevated using a microdissector and a fine rongeur. The dura was opened widely. At this point, the basilar artery and several branches could be clearly identified along with the major venous structures of the region (Figure 1). The anterior inferior cerebellar arteries could be clearly seen in all rats. In four rats, the dissection was continued rostrally beyond the suture line, exposing the sphenoid bone. Careful removal of the sphenoid bone exposed the pituitary fossa and the surrounding venous sinuses. The arachnoid overlying the basilar artery was opened.

Results Single-point or two-point BAO reduced peak-to-peak amplitude of the cortical SEPs by >50% within 15 minutes The SEPs gradually recovered over 3-4 hours, 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 No infarct in any rat with single-point basilar artery occlusion Two-point occlusion above or below the AICA produced brain stem infarcts Two-point BAO below AICA Two-point BAO above AICA

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%.

Parabrochial nucleus: It is located at the junction of the midbrain and pons in the lateral reticular formation, rostral to the parvocellular reticular nucleus near the superior cerebellar peduncle Pre-locus coeruleus is a small nucleus in the brainstem Parabrochial-precoeruleus

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. Noradreneric locus coeruleus Serotoninergic dorsal pedunculopontine: is located in the brainstem, caudal to the substantia nigra containing cholinergic neurons, the pars compacta, and one containing mostly glutamatergic neurons Laterodorsal tegmental nuclei: The laterodorsal tegmental nucleus (or lateroposterior tegmental nucleus) is a nucleus situated in the brainstem, spanning the midbrain tegmentum and the pontine tegmentum. The laterodorsal tegmental nucleus (LDT) sends cholinergic (acetylcholine) projections to many subcortical and cortical structures, including the thalamus, hypothalamus, substantia nigra (dopamine neurons), ventral tegmental area (dopamine neurons), cortex Parabrachial nucleus: It is located at the junction of the midbrain and pons in the lateral reticular formation, rostral to the parvocellular reticular nucleus near the superior cerebellar peduncle The relative influence of the two branches of the arousal system has also not been resolved. The thalamic branch, which innervates the intralaminar, relay, and reticular nuclei, has been thought to play a critical role in regulating thalamo-cortical transmission and the electroencephalographic (EEG) activity associated with sleep and wakefulness. A second branch runs through the lateral hypothalamus and basal forebrain, where it is augmented by additional neurons that project directly to the cerebral cortex. parabrachial region in the rostral pons on wakefulness, as this is a key source of inputs to the forebrain components of arousal systems

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

Results Ibotenic acid induced lesions of the thalamus the medial(parataenial and mediodorsal), anterior (anterodorsal,anteroventral, anteromedial, and laterodorsal), ventral(ventroanterior, ventrolateral, ventroposterior, and posterior), and intralaminar and midline (reuniens, paraventricular,rhomboid, centromedial, centrolateral, paracentraland parafascicular) nuclei were all nearly completely eliminated. Only a small portion of the reticular nucleus around the rim of the thalamus and the far caudal parts of the posterior and the medial and lateral geniculate nuclei were spared g: tyrosine hydroxylase

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

Figure 4. Normalized power spectra across 12 hours during either the light period (a) or the dark period (b) in control rats and rats with lesions of the thalamus (Tha), basal forebrain (BF), or parabrachial nucleus (PB). Note that the thalamic lesions caused only a loss of theta power, which was most marked during the dark period. The basal forebrain and parabrachial lesions caused extensive loss of EEG activity above the frequency of 1 Hz, with very little Remainin g EEG power above 4 Hz (i.e., above the delta range). Power spectrum: how the variance of the data is distributed over the frequency components into which may be decomposed 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 Figure 5. Effects of nonselective lesions of the BF on the EEG pattern and Fos expression induced by continuous stimulation (gentle touching). a1–a4: A series of sections (stained immunohistochemically, brown, for ChAT, with a blue thionin counterstain) arranged in rostro caudal order through the BF in a rat with bilateral lesions using orexin-saporin (125 ng total), which killed 88% of the cholinergic neurons and virtually all noncholinergic neurons in the BF. b: An enlarged view of the area in the red box in a3. Note that in the center of the lesion field (upper part of b), there are few if any surviving neurons remaining, whereas in the lower part of b, at the edge of the lesion area, about a dozen cholinergic cells (arrows) are the only surviving neurons in the field, which is otherwise filled with small glial nuclei. c: Neocortical Fos expression after 2 hours of sensory stimulation. d,e: A low level of Fos expression was seen in the neocortex, despite elevated Fos activity in the TMN (d) and the LC (e). f,g: EEG following BF ablation (i.e., during the coma-like state) demonstrated monotonous <1-Hz oscillation across all behaviors. f shows 12 seconds of EEG (at the time indicated by the arrow in h), and g shows a power spectrum for this 12-second period. Note that the only peak is in the sub-delta range. h: 20 minutes of EEG/EMG in this behaviorally unresponsive state. Note that the delta power (green) remains uniformly high and the theta power (magenta) low, even during brief abortive movements (spikes in the EMG trace). For abbreviations, see list. Scale bar . 2 mm in a1 (applies to a1–a4); 100 lM in b,d,e; 200 lM in c; 2 seconds (horizontal) and 50 lV (vertical) in f.

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

Effects of BF lesions on the EEG pattern induced by continuous stimulation f shows 12 seconds of EEG (at the time indicated by the arrow in h), and g shows a power spectrum for this 12-second period. Note that the only peak is in the sub-delta range. h: 20 minutes of EEG/EMG in this behaviorally unresponsive state. Note that the delta power (green) remains uniformly high and the theta power (magenta) low, even during brief abortive movements (spikes in the EMG trace). For abbreviations, see list. Scale bar ¼ 2 mm in a1 (applies to a1–a4); 100 lM in b,d,e; 200 lM in c; 2 seconds (horizontal) and 50 lV (vertical) in f. By continuous gentle touch, rats maintained a tonically active EMG. EEG showed a monotonous slow-wave activity

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

Summary 2 BF is a critical relay for maintaining the waking pattern of behavior, EEG and cFos expression. 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 Figure 9. Forebrain projections of the parabrachial nucleus (PB) and precoeruleus area (PC). a: The retrograde tracer CTB (stained brown immunohistochemically) is shown in the substantia innominata (SI; see inset), and retrogradely labeled neurons (brown) are seen in both the PC and the adjacent medial PB (marked by arrows). b: Double labeling with VGLUT2 mRNA radioisotopic in situ hybridization (black silver grains), after an injection of CTB into the SI (brown cells) shows that most neurons in the PB/PC that project to the BF express VGLUT2 mRNA (arrows). The inset shows an enlargement of two doubly labeled neurons marked by arrows just below the box. c: An illustration of the distribution of the CTB-labeled cells in the dorsolateral pontine region after an SI injection. Sections c1–c3 are arranged from rostral to caudal. Each dot . 3 cells. For abbreviations, see list. Scale bar . 100 lM in a; 50 lM in b; 200 lM in c.

PB/PC lesions induced by OX-SAP injection Figure 10. Effects of cell-selective lesions of the dorsolateral pontine tegmentum on sleep and wakefulness. a: OX-SAP lesions of the MPB caused an increase in the amount of both NREM and REM sleep during the dark period (Table 2; see also Lu et al., 2006b). b: Lesions involving both the MPB and the PC also showed loss of theta power in the EEG during REM sleep (see Lu et al., 2006). c–f: Larger lesions involving both the PC as well as the entire PB (c) caused coma with failure of continuous stimulation to activate Fos expression in the cerebral cortex (d) and reduced activation of the TMN (e; cf. Fig. 8c or g), although Fos expression in the LC (f) was elevated. g–l: Comparison of the physiology of a normal sleep-wake cycle (g–i) with the coma-like state (j–l). g shows a representative 12-second EEG epoch, and h shows the associated power spectrum (epoch begins at red arrow shown in the EEG trace from i) from an intact rat during NREM sleep. i shows the EEG (top trace) and EMG (bottom trace) during a period of NREM sleep, then REM sleep and then wake. The middle trace demonstrates the relative magnitude and changes in delta (d; green trace) and theta (y; magenta trace) power during this recording window. j,k: Representative 12-second EEG epoch (j) and associated power spectrum (k; epoch begins at red arrow shown in the EEG trace from l) from the same rat following a PB-PC lesion and the development of a ‘‘coma-like’’ state. The EEG and power spectrum from the behaviorally unresponsive animal clearly shows the predominantly <1-Hz sub-delta activity and loss of theta. Note in l that the EEG is monotonous in range, with prominent delta activity throughout the trace, and low EMG activity indicating lack of spontaneous movement. Total window time for traces in i and l was about 30 minutes (see time bar below l, which equals 2 minutes). For abbreviations, see list. Scale bar . 200 lM in a–d; 100 lM in e,f; 80 lV (vertical) in g; 50 lV (vertical) in j; 1 second (horizontal) in g,j. 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 g–l: Comparison of the physiology of a normal sleep-wake cycle (g–i) with the coma-like state (j–l). g shows a representative 12-second EEG epoch, and h shows the associated power spectrum (epoch begins at red arrow shown in the EEG trace from i) from an intact rat during NREM sleep. i shows the EEG (top trace) and EMG (bottom trace) during a period of NREM sleep, then REM sleep and then wake. The middle trace demonstrates the relative magnitude and changes in delta (d; green trace) and theta (y; magenta trace) power during this recording window. j,k: Representative 12-second EEG epoch (j) and associated power spectrum (k; epoch begins at red arrow shown in the EEG trace from l) from the same rat following a PB-PC lesion and the development of a ‘‘coma-like’’ state. The EEG and power spectrum from the behaviorally unresponsive animal clearly shows the predominantly <1-Hz sub-delta activity and loss of theta. Note in l that the EEG is monotonous in range, with prominent delta activity throughout the trace, and low EMG activity indicating lack of spontaneous movement. Total window time for traces in i and l was about 30 minutes (see time bar below l, which equals 2 minutes). By continuous gentle touch, rats maintained a tonically active EMG. EEG showed a monotonous slow-wave activity

Summary 3 PB/PC is critical for achieving and maintaining an activated EEG and a waking state. 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.

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