Download Brain Cell Death Is Reduced With Cooling by 3.5°C to 5°C

Brain Cell Death Is Reduced With Cooling by
3.5°C to 5°C but Increased With Cooling by 8.5°C in
a Piglet Asphyxia Model
Daniel Alonso-Alconada, PhD; Kevin D. Broad, PhD; Alan Bainbridge, PhD;
Manigandan Chandrasekaran, MD; Stuart D. Faulkner, PhD; Áron Kerenyi, MD;
Jane Hassell, MBBS; Eridan Rocha-Ferreira, PhD; Mariya Hristova, PhD; Bobbi Fleiss, PhD;
Kate Bennett, MSc; Dorottya Kelen, MD; Ernest Cady, FInstP, FIPEM; Pierre Gressens, PhD;
Xavier Golay, PhD; Nicola J. Robertson, MB ChB, PhD
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Background and Purpose—In infants with moderate to severe neonatal encephalopathy, whole-body cooling at 33°C to
34°C for 72 hours is standard care with a number needed to treat to prevent a adverse outcome of 6 to 7. The precise brain
temperature providing optimal neuroprotection is unknown.
Methods—After a quantified global cerebral hypoxic-ischemic insult, 28 piglets aged <24 hours were randomized (each group, n=7)
to (1) normothermia (38.5°C throughout) or whole-body cooling 2 to 26 hours after insult to (2) 35°C, (3) 33.5°C, or (4) 30°C.
At 48 hours after hypoxia-ischemia, delayed cell death (terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick
end labeling and cleaved caspase 3) and microglial ramification (ionized calcium-binding adapter molecule 1 ) were evaluated.
Results—At 48 hours after hypoxia-ischemia, substantial cerebral injury was found in the normothermia and 30°C hypothermia
groups. However, with 35°C and 33.5°C cooling, a clear reduction in delayed cell death and microglial activation was
observed in most brain regions (P<0.05), with no differences between 35°C and 33.5°C cooling groups. A protective pattern
was observed, with U-shaped temperature dependence in delayed cell death in periventricular white matter, caudate nucleus,
putamen, hippocampus, and thalamus. A microglial activation pattern was also seen, with inverted U-shaped temperature
dependence in periventricular white matter, caudate nucleus, internal capsule, and hippocampus (all P<0.05).
Conclusions—Cooling to 35°C (an absolute drop of 3.5°C as in therapeutic hypothermia protocols) or to 33.5°C provided
protection in most brain regions after a cerebral hypoxic-ischemic insult in the newborn piglet. Although the relatively
wide therapeutic range of a 3.5°C to 5°C drop in temperature reassured, overcooling (an 8.5°C drop) was clearly
detrimental in some brain regions. (Stroke. 2015;46:275-278. DOI: 10.1161/STROKEAHA.114.007330.)
Key Words: hypothermia ◼ hypoxia-ischemia, brain ◼ neonatal encephalopathy ◼ neuroprotection
T
herapeutic hypothermia is now standard clinical care for
moderate to severe neonatal encephalopathy in the United
Kingdom and developed world.1 Clinical trials have included
whole-body cooling with core temperature reduced to 33.5°C for
72 hours2 because the optimal temperature for neural rescue is
likely to be below 34°C. There are, however, 40–50% of infants
who, despite hypothermic treatment, have an adverse neurodevelopmental outcome.1 Cooling to lower temperatures has been
suggested, particularly for those with severe encephalopathy,3 but
the ideal temperature for brain protection is unknown.
The aim of the current study was to assess brain regional
cell death and microglial activation under normothermia, and
with cooling to 35°C, 33.5°C, and 30°C from 2 to 26 hours
after a global cerebral hypoxic-ischemic insult in the piglet
asphyxia model.
Methods
Expanded Methods are available in the online-only Data Supplement.
All experimentation was under UK Home Office Guidelines
(Animals [Scientific Procedures] Act 1986) and approved by the
Animal Care and Use Committee of University College London
Biological Services and Institute of Neurology.
The hypoxic-ischemic insult was performed in 28 large white male
piglets aged <24 hours as previously described.4 After hypoxia-ischemia
(HI) and resuscitation, piglets were randomized into 4 groups: (1) normothermia (rectal temperature [Trec], 38.5°C throughout) or whole-body
cooling 2 to 26 hours after insult to Trec (2) 35°C, (3) 33.5°C, or (4) 30°C
Received September 20, 2014; final revision received October 25, 2014; accepted October 28, 2014.
From the Institute for Women’s Health, University College London, London, United Kingdom (D.A.-A., K.D.B., M.C., S.D.F., A.K., J.H., E.R.-F.,
M.H., K.B., D.K., N.J.R.); Medical Physics and Bio-engineering, University College London Hospitals NHS Foundation Trust, London, United Kingdom
(A.B., E.C.); Centre for the Developing Brain, King’s College London, London, United Kingdom (B.F., P.G.); and Department of Brain Repair and
Rehabilitation, Institute for Neurology, Queen Square, London, United Kingdom (X.G.).
The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.114.
007330/-/DC1.
Correspondence to Nicola J. Robertson, MB ChB, PhD, Perinatal Neuroscience and Honorary Consultant Neonatologist, Institute for Women’s Health,
University College London, 74 Huntley St, London WC1E 6HX, United Kingdom. E-mail [email protected]
© 2014 American Heart Association, Inc.
Stroke is available at http://stroke.ahajournals.org
DOI: 10.1161/STROKEAHA.114.007330
275
276 Stroke January 2015
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Figure 1. Piglet brain regions assessed for immunohistochemistry. (1) Dorsal cortex; (2) motor/visual cortex; (3) somatosensory
cortex; (4) midtemporal cortex; (5) pyriform area; (6) periventricular white matter; (7) caudate; (8) internal capsule; (9) putamen;
(10) hippocampus; and (11) thalamus.
(all n=7). Normothermic piglets were maintained at their target Trec using a warmed water mattress above and below the animal; hypothermia
piglets were cooled (by reducing the water mattress temperature) to their
target Trec over 90 minutes starting 2 hours after HI. At 26 hours after HI,
cooled piglets were rewarmed to normothermia at 0.5°C/h using a water
mattress with circulating water heated to increasing temperatures.
At 48 hours after HI, piglets were euthanized and 11 brain regions
(Figure 1) were analyzed for histology and immunohistochemistry
(online-only Data Supplement).
Results
There were no differences in body weight, postnatal age,
insult severity, or baseline physiological and biochemical
measures (data not shown) between the temperature groups.
The physiological and systemic effects of cooling to different
temperatures have been previously described in detail4 (Table
I in the online-only Data Supplement).
As shown in Figure 2, there was a reduction in the number
of terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling positive cells in the 35°C and 33.5°C
hypothermia groups when compared with the normothermic
group, an effect being absent at 30°C. Figure 3 (extended in
Table II in the online-only Data Supplement) shows that cooling at 33.5°C and 35°C resulted in lower terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling
counts (compared with normothermia) in midtemporal cortex,
periventricular white matter, caudate, putamen, hippocampus,
and thalamus, with no differences between both cooling groups.
Cooling at 30°C reduced cell death only in the hippocampus.
Cooling at 33.5°C reduced cleaved caspase 3–positive cells
in hippocampus and thalamus in comparison with normothermia (Figure 3; extended in Table III in the online-only Data
Supplement). Cooling at 30°C was not associated with lower
cleaved caspase 3 counts. In the periventricular white matter,
the numbers of cleaved caspase 3–positive cells were reduced
in all temperature groups.
HI after normothermia resulted in the loss of the microglial
branches, with many cells transforming into completely rounded
brain macrophages (Figure 2, bottom). When compared with normothermia, cooling at 35°C or 33.5°C significantly preserved ramification index, showing a similar increase in ramification with no
difference between groups. The 30°C group showed a lower ramification index than the 35°C and 33.5°C groups in most regions.
Discussion
This study demonstrated substantial brain injury after HI in
newborn piglets subsequently maintained normothermic and
also in those treated with 30°C hypothermia, but a clear reduction in cell damage by cooling to 35°C and 33.5°C. Moreover,
a temperature-dependent protective pattern was observed in
some brain regions, with U-shaped temperature dependence
for delayed cell death and inverted U-shaped temperature
dependence for microglial ramification.
Figure 2. Representative photomicrographs from each temperature group at
48h after hypoxia-ischemia. Top, TUNELpositive cells (thalamus). Middle, Cleaved
caspase-3–positive cells (thalamus). Bottom, Ionized calcium-binding adapter molecule 1 (Iba-1) immunostaining (caudate).
Alonso-Alconada et al Therapeutic Hypothermia After Perinatal Asphyxia 277
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Figure 3. TUNEL, cleaved caspase 3–positive
cells, and microglial ramification index at 48
hours after hypoxia-ischemia in 11 brain regions
according to temperature group. *P<0.05 vs normothermia and †P<0.05 vs 30°C. Cdt indicates
caudate; dCTX, dorsal cortex; Hip, hippocampus;
IBA-1, ionized calcium-binding adapter molecule 1;
IC, internal capsule; mCTX, midtemporal cortex;
mvCTX, motor/visual cortex; Ptmn, putamen;
PvWM, periventricular white matter; Pyr, pyriform
area; sCTX, somatosensory cortex; and Thal,
thalamus.
Apart from the commonly known decrease in the metabolic
rate (7%–9% per 1°C core temperature reduction) with parallel decreases in O2 consumption and CO2 production, the beneficial effects of hypothermia include reduced excitotoxicity,
calcium antagonism, protein synthesis preservation, decreased
edema, modulation of the inflammatory cascade, and a change
in proapoptotic and antiapoptotic signaling.5 However, hypothermia may also have adverse effects, such as reduced cardiac
contractility, reduced cerebral blood flow, poor perfusion, sympathetic and neuroendocrine stimulation, and increased blood
viscosity.6–8 It is likely, therefore, that an effective temperature
range exists below which hypothermic neuroprotection is lost;
this therapeutic temperature range may be influenced by severity, the delay in onset and duration of cooling, and other factors, such as the peripheral immune response.9
Cooling from 38.5°C to 30°C (an 8.5°C absolute temperature drop) neither reduced delayed cell death nor maintained
the microglial ramification index in most of the brain regions
evaluated, showing a global neuropathological pattern similar to that for normothermia. These histological results accord
278 Stroke January 2015
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with physiological observations previously documented by our
group,4 describing abnormal metabolic homeostasis with lactic
acidosis, hyperglycemia, hypokalemia, and an increased need
for inotrope and fluid bolus support to maintain the mean arterial
blood pressure for 30°C cooling compared with other temperatures. Cooling from 38.5°C to 35°C or 33.5°C, ie a reduction of
3.5°C to 5°C, was associated with a similar profile of protection
based on terminal deoxynucleotidyl transferase deoxyuridine
triphosphate nick end labeling staining, with no difference in the
number of terminal deoxynucleotidyl transferase deoxyuridine
triphosphate nick end labeling positive cells between the 35°C
and 33.5°C cooling groups. For caspase positive cells, although
a significant difference was seen for more regions after cooling
to 33.5°C (hippocampus and thalamus), this must be taken in
the context that caspase expression was seen in a subset of dead
cells only and, therefore, the apparent difference may represent a
change in the speed of evolution of death, not total death. These
data may suggest that the extra 1.5°C drop in temperature is
unlikely to be harmful, thus helping to explain why therapeutic
hypothermia has been successful, despite considerable variation in the stringency of temperature control between clinical
trials. However, some caution is needed because the Optimizing
Cooling trial, which compared cooling deeper (32°C) and longer
(120 hours) with conventional cooling protocols, was recently
closed for safety and futility after 364 of planned 726 infants
were enrolled after recommendation from the data and safety
monitoring committee (http://clinicaltrials.gov/ct2/show/results/
NCT01192776). The in-hospital mortality rate increased from
7% to 14%, when 72-hour cooling at 33.5°C and 32°C was compared, suggesting that cooling deeper could be harmful.10
Although the extent of cell death and tissue damage varies according to brain region, the overall neuroprotective effect
of hypothermia is determined by the local tissue susceptibility to injury. In this study, cooling to 30°C led to worse injury
in some brain areas, such as thalamus, putamen, and caudate
nucleus; these data show a U-shaped temperature dependence of
reduced delayed cell death and an inverted U-shaped temperature
dependence of maintained microglial ramification. Previously
described harmful side effects of hypothermia combined with the
intrinsic cerebral metabolic features of the deep gray matter may
render deep gray matter more vulnerable to injury from both HI
and overzealous cooling. These peripheral effects are likely to
occur to a different extent in neonatal encephalopathy and will
depend on the extent of multiorgan failure and severity of HI.
It must be taken into consideration that the relative decrease
in temperature in the present study was greater in our piglets
(normal core temperature, 38.5°C) than would be the case for
the same target core temperature in the human infant; relatively large temperature reduction and metabolic rate reduction
were, therefore, induced in our piglet studies compared with
encephalopathic human newborns cooled to 33.5°C. There are
other important differences between species; for example, piglets have a higher basal cerebral metabolic rate of oxygen11 and
greater metabolic rate reduction with hypothermia12 than human
newborns. Whether it is better to induce the actual cooling temperature used in existing clinical protocols in human infants or
induce the clinical temperature drop from the normothermic
piglet temperature for these translational studies is unclear. Our
cooling protocol (24 hours of hypothermia duration) is shorter
than the current clinical protocol (72 hours of cooling),2 and further cell death may occur beyond the 48-hour time-point when
we quantified it; nevertheless, several long-term studies have
previously demonstrated a permanent protective effect.13,14
This piglet asphyxia study of cooling from 2 to 26 hours
after a global hypoxic-ischemic insult demonstrates that optimal neuronal and white matter protection was seen with a
reduction in the core temperature of 3.5°C to 5°C (35°C and
33.5°C, respectively). Although the relatively wide therapeutic range of 1.5°C is reassuring, these data emphasize the
potential detrimental effects of excessive cooling.
Acknowledgments
This work was undertaken at University College London Hospitals/
University College London.
Sources of Funding
United Kingdom Medical Research Council (G0501259), Basque
Government Postdoctoral Program (POS_2013_1_191), and United
Kingdom Department of Health’s National Institute of Health
Research Biomedical Research Centres Funding Scheme.
Disclosures
None.
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Brain Cell Death Is Reduced With Cooling by 3.5°C to 5°C but Increased With Cooling by
8.5°C in a Piglet Asphyxia Model
Daniel Alonso-Alconada, Kevin D. Broad, Alan Bainbridge, Manigandan Chandrasekaran,
Stuart D. Faulkner, Áron Kerenyi, Jane Hassell, Eridan Rocha-Ferreira, Mariya Hristova, Bobbi
Fleiss, Kate Bennett, Dorottya Kelen, Ernest Cady, Pierre Gressens, Xavier Golay and Nicola J.
Robertson
Stroke. 2015;46:275-278; originally published online November 25, 2014;
doi: 10.1161/STROKEAHA.114.007330
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