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T3 replacement does not prevent excitotoxic cell death but reduces developmental neuronal apoptosis in newborn mice
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Official Journal of the European Paediatric Neurology Society
Original article
T3 replacement does not prevent excitotoxic cell death but reduces developmental neuronal apoptosis in newborn mice Gergely Sa´rko¨zya,b,1, Elke Griesmaiera, Xiangying Hea,c, Klaus Kapelaria, Martina Urbaneka, Georg Simbrunera, Pierre Gressensd, Matthias Kellera,,1 a
Department of Paediatrics IV, Neonatology, Neuropediatrics and metabolic diseases, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck, Austria b 2nd Department of Paediatrics, Semmelweis University Budapest, Hungary c Department of Pediatrics, The Third Affiliated Hospital of Zhengzhou University, PR China d INSERM U 676 and Service de Neurologie Pe´diatrique, Hoˆpital Robert Debre´ 48, Blvd Serurier, F-75019 Paris, France
art i cle info
ab st rac t
Article history: Received 25 September 2006
Background: Periventricular leukomalacia (PVL) is a major cause of neurological handicap
Received in revised form
in pre-term infants. At present, there are no effective or causal therapies available. Thyroid
26 November 2006
hormones play an essential role in brain development and are reported to be decreased in
Keywords:
Hypothesis: Excitotoxic brain damage of newborn mice decreases thyroid hormone
pre-terms and following brain injury in adults.
Brain injury Newborn Excitotoxicity T3
concentrations. Exogenous T3 administration restores thyroid hormone levels and reduces perinatal brain damage in an animal model of PVL.
Design and method: To create white and gray matter (WM/GM) lesion mimicking several key aspects of PVL, we injected ibotenic acid (Ibo), a glutamate analog, into the right hemisphere (intracranially (i.c.)) of 5-day-old mice. T3 (10 mg/kg body weight (bw)) was injected intraperitoneally (i.p.) 1 h or repeatedly 1/24/48/72/96 h post-insult. We determined lesion size, number of apoptotic cells in WM/GM and serum T3/T4 concentration at 24 and 120 h after injury. Serum T3/T4 concentration was also determined before and 1 and 2 h after T3 administration.
Results: Excitotoxic brain damage did not alter serum T3/T4 concentrations within 120 h of injury. Serum T3 levels were distinctly elevated within 1 h of T3 injection; however, this elevation was relatively short-lived (half-life estimated to be less than 12 h). Neither single nor repetitive T3 treatment regimen reduced excitotoxic lesion size, but it did reduce apoptosis.
Corresponding author. Tel.: +43 512 504 81567; fax: +43 512 504 27766.
E-mail address: [email protected] (M. Keller).
abbreviations: bw, body weight; T3, Triiodthyronine; T4, thyroxine; i.c., intracranial; i.p., intraperitoneal; PBS, phosphatebuffered saline; PVL, periventricular leukomalacia 1 Both authors contributed to the same part. 1090-3798/$ - see front matter & 2006 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ejpn.2006.11.009
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Conclusions: T3 replacement does not prevent excitotoxic cell death, but it does reduce developmental neuronal apoptosis, which could participate to the beneficial neuropsychological effects of hormone therapy. Further study is therefore warranted. & 2006 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
Pre-term infants are at high risk for brain damage and subsequent neurological sequelae. Depending on their birth weight, 20–50% of these infants suffer severe motor disabilities and cognitive deficits.1,2 Aside from the socioeconomic effects and high costs to the health care system, the adverse neurological outcome imposes a severe burden to both the child and parents. Damage to white matter (WM), including periventricular leukomalacia (PVL), is one of the most common brain injury occurring in pre-term infants. Inflammation,3,4 hypoxia/ischemia, free oxygen radical formation5 and excitotoxicity6–8 are all pathogenic mechanisms known to mediate this injury. Additionally, a sudden decline in hormone levels (for instance cortisol, vasoactive intestinale polypeptide and thyroxine) is currently considered to be a risk factor for brain damage and increased mortality in pre-term infants.9,10 Very early pre-term infants are deficient in thyroid hormones because (i) premature infants suddenly lack their mother’s thyroid and (ii) because their hypothalamo–pituitary–thyroid and thyroid metabolism systems have not matured at this developmental stage of life. The result is a period of transient hypothyroxinemia, during which bound and free plasma concentrations of thyroxine (T4) and triiodothyronine (T3) are low.11 In several cohort studies, very low concentrations of T3 or T4 are associated with an increased risk of impaired developmental outcome.11–13 Further, low serum thyroxine, as determined by initial newborn screening, was associated with intraventricular hemorrhage and higher mortality in very low birth weight infants.14,15 After adjusting for potential confounders, such as low gestational age and measures of illness severity, premature infants with hypothyroxinemia had twice the risk of developing sonographic echolucency (indicating PVL) than their peers with higher thyroxine levels.16 It was therefore speculated that the additional T3/T4 substitution might improve neurological and neurodevelopmental outcome in pre-term infants. Several clinical trials have assessed whether exogenous T3/T4 administration improves neurological outcome of pre-terms17,18; however, the results remain controversial.19 Of note, subgroup analysis comparing infants with and without brain damage was not possible in these clinical trials, and thus a protective effect of T3/T4 administration could have been overlooked. Although a matter of particular interest, it is not known whether low T3/T4 values are associated with brain damage (i.e., ‘‘markers’’ of damage), or whether they possess a causal relationship (i.e., ‘‘inducers’’ of damage). In adults, brain damage per se is known to decrease T3 levels20; whether this happens also in newborns has not been investigated.
On this basis, we hypothesized (i) that excitotoxic brain damage decreases total T3/T4 levels in serum; and (ii) that exogenous systemic administration of T3 is neuroprotective against NMDA receptor-mediated brain injury in an animal model of excitotoxic perinatal brain damage mimicking several key aspects of PVL.
2.
Materials and methods
2.1.
Materials
Ibotenic acid (Ibo) tained from Biotrend (Germany), phosphate-buffered saline (PBS) from Invitrogen (Austria) and 3,30 ,5-triiodo-L-thyronine sodium salt from Sigma (Saint Louis, Missouri, USA). Test-kits and reagents: anti-cleaved caspase-3 antibody (Cell Signaling, New England Biolabs, Germany), biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA), streptavidin–biotin complex (Vectastain ABC kit; Vector Laboratories, Burlingame, CA, USA), Immu-Mount (Thermo-Shandon), Diaminobenzidine (Fluka, Buchs, Switzerland).
2.2.
Animal model of excitotoxic brain injury
We used the animal model of excitotoxic brain damage as described previously.21 Briefly 5-day-old CD1 mice were bred at the animal facility of the Medical University of Innsbruck, weighed, and mice with a body weight of 370.3 g at day P5 were included in this study. They were housed under standard conditions in a conventional animal facility with their dams. Adequate measures were taken to minimize pain or discomfort, complying with the European Community guidelines for the use of experimental animals. All pups investigated were anesthetized with isoflurane and kept under a warming lamp. Intracranial (i.c.) injections were performed with a 26-G needle on a 50 ml Hamilton syringe mounted on a calibrated micro-dispenser. The needle was inserted stereotactically 2 mm under the external surface of the scalp in the fronto-parietal area of the right hemisphere, 2 mm from the midline in the lateral–medial plane and 3 mm from the junction between the sagittal and lambdoid sutures in the rostro-caudal plane. Two 1 ml boluses of Ibo (5 mg/ml) were injected 2 and 1 mm under the surface 20 s apart. The needle was left in place for 20 s after the second bolus to avoid leakage. This produces a lesion in WM that mimics the PVL observed in pre-term neonates and striatal/cortical plate injury in full-term human infants. The development of the lesion in this model can be blocked by treatment with NMDA receptor antagonist.21 After injection, the animals were returned to their dams. Histopathology confirmed that the tip of the needle had always reached the periventricular WM.
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2.3.
Experimental groups and study design
Animals from at least four separate litters received either no insult (control) or an i.c. injection of ibotenate (10 mg/2 ml) on post-natal day 5 to create WM and GM lesions similar to those observed in human PVL. The animals of these two groups were further subdivided into five treatment groups: (1) no i.c. or intraperitoneal (i.p.) injections; (2) a control group of one single i.p. injection of vehicle (PBS); (3) a treatment group of a single i.p. injection of T3 (10 mg/kg bodyweight (bw)); (4) a control group of 5 i.p. injections of PBS; and (5) a treatment group of 5 i.p. T3 injections (10 mg/kg bw). Groups 2 and 3 received their PBS or T3 administration 1 h post-injection of ibotenate, while groups 4 and 5 received their PBS or T3 administrations at 1, 24, 48, 72 and 96 h following ibotenate injection. Groups 2 and 3 were analyzed with respect to lesion size, apoptosis, and T3/T4 serum concentrations 24 h following lesion setting, while groups 4 and 5 were analyzed after 120 h. The absolute dose of T3 was adapted daily to the current body weight.
2.4.
Lesion size determination
Mouse pups were euthanized by decapitation either 24 or 120 h following the excitotoxic injury. Brains were immediately immersed in 4% formaldehyde and fixed for 72 h. Following paraffin embedding, blocks were sliced into 10 mm coronal sections. Every third section was stained with cresyl violet and the lesion size was determined as described elsewhere.21,22 Briefly, neocortical and WM lesions can be defined by the maximal length of three orthogonal axes: a horizontal and radial axis in the coronal plane and one in the fronto-occipital axis (in a sagittal plane). In previous studies, we have shown an excellent correlation between the maximum size of the different diameters and excitotoxic lesions. Based on these observations, we serially sectioned the entire brain in the coronal plane. This permitted an accurate and reproducible determination of the maximum sagittal frontooccipital diameter (which is equal to the number of sections where the lesion was present multiplied by 10 mm) and this was used as an index of the volume of the lesion. Two observers independently determined lesion size, each blinded to the treatment groups being analyzed. Data are presented as mean length of the lesion in the rostro-caudal axis7SEM.
2.5.
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streptavidin–biotin complex (Vectastain ABC kit; Vector Laboratories, Burlingame, CA, USA). Visualization was performed using diaminobenzidine as the chromogen. Following multiple rinses in distilled water, sections were dehydrated, cleared in xylene and coverslipped. Sections from similar anatomical areas were processed simultaneously. Labeled cells in the neocortical layers and underlying WM at the level of ibotenate-induced lesions (edges and maximum of the lesion) in the injured and noninjured hemispheres, as well as in the basal ganglia were counted by one observer, who was blinded to the treatment group. Six brains from each group were randomly selected, and 8–12 brain sections of 10 mm thickness were examined from each brain. Data are given as mean number of positive cells in WM and GM per brain7SEM.
2.6.
Determination of T3 and T4 in serum
To assess the effect of brain damage, and of single and repetitive T3 administration on total T3 and T4 serum concentration in newborn mice, we determined T3 and T4 serum concentration in (1) healthy mice with no intervention; (2) brain injured mice (i.c. injection of ibotenate)+PBS i.p.; (3) brain injured mice (i.c. injection of ibotenate)+one single T3 i.p. injection; and (4) brain injured mice (i.c. injection of ibotenate)+repetitive T3 i.p. injections. Measurement of serum T3/T4 concentrations was completed on days P5, P6 and P10 for group 1; days P6 and P10 for group 2; day P6 for group 3 and day P10 for group 4. Determination of total serum T3/T4 from blood samples was performed using an electrochemiluminescence immunoassay (ELICA) for quantification (Roche, Mannheim, Germany) according to the manufacturer’s instructions.
2.7.
Statistics
Two standard statistical tests were employed in this study. An unpaired Student’s t-test was employed when only two experimental groups were compared. For multiple comparisons, and ANOVA was first employed to determine if any of the treatment groups statistically differed, followed by t-tests with Dunnet’s correction in order to determine which groups differed. Statistical differences were considered to be significant at a level of po0.05.
Immunohistochemistry for cleaved caspase-3
3. Sections (10 mm) of formaldehyde-fixed and paraffin-embedded mouse brain were deparaffinized in xylene and passed through graded alcohols. Endogenous peroxidase activity was quenched by incubation in 2% hydrogen peroxide in methanol for 30 min. Heat-induced antigen retrieval was performed using 10 mM citrate buffer (pH 6.0), for 15 min in a microwave oven. After blocking with 10% fetal calf serum (in PBS), sections were exposed to a rabbit anti-cleaved caspase-3 (Asp 175) antibody (1:500; Cell Signaling Technology via New England Biolabs GmbH, Frankfurt, Germany). The sections were subsequently incubated for 45 min at 25 1C with biotinylated goat anti-rabbit IgG (1:100; Vector Laboratories, Burlingame, CA, USA) and then for 45 min at 25 1C with a
Results
3.1. Excitotoxic brain damage does not decrease total T3/ T4 serum concentrations in newborn mice As displayed in Fig. 1, excitotoxic brain damage is not associated with a decrease in serum T3 and T4 concentrations (n ¼ 3–7), at least within 120 h of the initial insult. In control (untreated) animals, we observed that T3 levels significantly decreased (Fig. 1a) and T4 levels significantly increased (Fig. 1b) between days P6 and P10. The serum T3/T4 levels in the two excitotoxic injury treatment groups (Ibo+PBS/Ibo+T3) were not different when compared to their time-matched controls.
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a
24 h post-insult (P6) and 120 h post-insult (P10). In the case of the latter, single and repetitive T3 injection treatments were compared. There were no significant differences in lesion size when the treatment groups were compared to their timematched control. Although there were tendencies for the lesion sizes to change between day P6 and P10 (smaller for WM; larger for GM), these differences were not found to be significant.
+ single/5 x PBS i.p.
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Ibo i.c. + single/ 5 x PBS i.p. Ibo i.c. + single/5 x T3 i.p.
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3.3. Exogenous T3 decreases developmental apoptosis in the newborn brain
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T4 (μg/dl)
6
4.
Discussion
4
2
n=3
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0 P5
P6
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Fig. 1 – Quantitative effects of age, excitotoxic brain damage and T3 supplementation on total T3 (a) and T4 (b) concentrations in serum. Animals were treated with a single dose (P6) or with 5 repetitive doses (beginning on P6, every 24 h until P10) of T3 or PBS respectively. Excitotoxic lesions were produced on P5 and serum T3 and T4 concentrations were analyzed on days P5, P6 and P10. Bars represent mean concentration of T3 and T47SEM. Numbers in the bars represent the number of animals used in each experimental group (n ¼ 3–7).
Notably, a single injection of T3 (10 mg/kg bw), administered 1 h post-excitotoxic insult, did not cause alterations in T3/T4 levels measured at 24 (P6) and 120 (P10) h post-insult. The T3 injections, therefore, result in only a transient elevation in T3 levels, which were observed within 1–2 h of the T3 injection (pre-T3 injection: 145712 ng/dl; 1 h post-T3 injection: 1410790 ng/dl, po0.01 compared to pre-injection; 2 h postT3 injection: 1356798 ng/dl, po0.01 compared to pre-injection).
3.2.
As shown in Fig. 3, repetitive T3 treatment significantly reduced apoptosis. Excitoxicity per se slightly increased the number of cells positive for caspase-3 cleavage (a marker of apoptosis) at 120 h after the insult in GM and WM compared to the undamaged hemisphere. In both, the damaged (Fig. 3 right), but also in the undamaged (Fig. 3 left) hemispheres the number of cells positive for caspase-3 cleavage was significantly reduced in animals treated with T3.
I.p. T3 does not reduce excitotoxic brain injury
Fig. 2 shows that following excitotoxic insult, injection of T3 (10 mg/kg bw) does not reduce WM (Fig. 2a and b) or GM (Fig. 2a and c) lesions. Lesion size was assessed at two time points:
In the present study, we show that exogenous T3 administration does not decrease NMDA receptor-mediated excitotoxic brain damage in newborn mice. In contrast to our hypothesis, neither a single nor repetitive i.p. administrations of T3 reduced WM or GM lesion size. Furthermore, excitotoxic brain injury in newborn mice per se did not alter total T3 and T4 serum concentrations within 120 h of the insult. We systematically assessed the age-dependent physiological changes in T3 and T4 concentrations and found that both hormone levels changed between days P6 and P10. This highlights the need for similar studies to have the proper time-matched controls. Of note, exogenous T3 administration did not result in sustained changes in T3 or T4 levels (Fig. 1). The half-life of injected T3 is known to be relatively long in newborn humans (24 h), but it can be as short as 6 h in other newborn mammals, such as piglets.23 The half-life of T3 in mice is not known, but our data suggests that it is less than 12 h. The primary aim of the present study was to determine if exogenous T3 administration could reduce excitotoxic brain injury. The T3 dosage employed was within the range of doses used in other studies,24,25 and was sufficiently low enough that it did not cause hyperthyroidism (50 mg/kg bw26; i.p. injection of T3 resulted in a marked increase in T3 serums levels (within 1 h); previous studies indicate that T3 levels in venous blood reach almost all brain centers (including GM, cerebellum, caudate/putamen, hippocampal pyramidal layer, dentate gyrus, thalamic nuclei, superior and inferior colliculi, brain stem nuclei and ventricles) within 3 h.27 Injection of T3 did not ameliorate ibotenate-mediated excitotoxic cell death. However, since injected T3 appears to have a relatively short half-life in the bloodstream (less than 12 h), future studies should attempt a procedure that results in sustained T3 elevation. Administration of T4, for example,
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Fig. 2 – (a) Representative photographs (5 magnification) of brain slices stained with cresyl violet. The photographs display the cyst and cortical atrophy in the PBS- and T3-treated mice following excitotoxic injury. (b/c) Quantitative effect of T3 i.p. treatment on ibotenate-induced white (b) and gray matter (c) lesions. Lesions were produced on P5 and analyzed on days P6 and P10. Bars represent mean length of the lesions in the fronto-occipital axis 7SEM. Numbers in the bars represent the number of animals used in each experimental group (n ¼ 8–12).
may result in higher intracellular levels of T3 and may have a longer half-life. Such protocols, however, were beyond the scope of the present study. Injection of T3 was found to reduce developmental neuronal apoptosis. Neuronal apoptosis is a normal physiological process of brain development, the functional significance of which remains unclear (see Ref.28 for a review). The consequences of reducing (or delaying) the developmental apoptosis of neocortical neurons are unknown; whether it is beneficial or detrimental likely depends on physiological or patho-physiological state of the brain at the time of intervention. Behavioral studies will be necessary to determine the
functional correlates of this histological finding. However, there is a potential for T3 administration to be beneficial in pre-term infants at risk of neuronal loss. This is consistent with other clinical data indicating better schooling and motor outcomes in children who were supplemented with T4 following very early (o27–28 weeks) premature birth.29 On a cautionary note, however, the reverse was true when T4 was administered to slightly older infants of 29 weeks’ gestation.29 In our experimental setting, T3 supplementation did not alter the T4 serum concentration in newborn mice. However, we cannot exclude the possibility that T3 supplementation may result in a significant reduction in the serum T4 level of
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Fig. 3 – (a) Representative photographs, 20 magnification) of brain slices immuno-stained for activated caspase-3 (a marker of apoptosis) in PBS- and T3- and treated mice indicating the decrease in apoptosis in undamaged brain tissue by T3 treatment. (b) Quantitative effects of T3 (10 lg/kg) i.p. treatment on ibotenate-induced apoptosis in gray matter and the underlying white matter. Data are presented as number of cleaved caspase-3 positive cells 7SEM in the right (damaged) and left (undamaged) hemisphere at day P10. Asterisks indicate statistically significant differences from PBS-treated controls (po0.05, Student’s t-test). Numbers in the bars represent the number of animals used in each experimental group.
human pre-term infants, thereby resulting in a critical reduction in brain T3. In this context, T3 supplementation is not recommended until its safety is confirmed. In conclusion, exogenous T3 administration does not reduce excitotoxic cell death. T3 administration appears to reduce developmental neuronal apoptosis, which could participate to the beneficial neuropsychological effects of hormone therapy in pre-term infants less than 28 weeks of gestation.
Acknowledgment We thank Darcy Lidington for carefully proof reading this manuscript and his valuable contributions.
R E F E R E N C E S
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6. Loeliger M, Watson CS, Reynolds JD, Penning DH, Harding R, Bocking AD, et al. Extracellular glutamate levels and neuropathology in cerebral white matter following repeated umbilical cord occlusion in the near term fetal sheep 1. Neuroscience 2003;116:705–14. 7. Marret S, Mukendi R, Gadisseux JF, Gressens P, Evrard P. Effect of ibotenate on brain development: an excitotoxic mouse model of microgyria and posthypoxic-like lesions. J Neuropathol Exp Neurol 1995;54:358–70. 8. Volpe JJ. Neurobiology of periventricular leukomalacia in the premature infant. Pediatr Res 2001;50:553–62. 9. O’Shea TM. Cerebral palsy in very preterm infants: new epidemiological insights 12. Ment Retard Dev Disabil Res Rev 2002;8:135–45. 10. Zupan V, Nehlig A, Evrard P, Gressens P. Prenatal blockade of vasoactive intestinal peptide alters cell death and synaptic equipment in the murine neocortex. Pediatr Res 2000;47:53–63. 11. Lucas A, Morley R, Fewtrell MS. Low triiodothyronine concentration in preterm infants and subsequent intelligence quotient (IQ) at 8 year follow up. BMJ 1996;312:1132–3. 12. den Ouden AL, Kok JH, Verkerk PH, Brand R, VerlooveVanhorick SP. The relation between neonatal thyroxine levels and neurodevelopmental outcome at age 5 and 9 years in a national cohort of very preterm and/or very low birth weight infants. Pediatr Res 1996;39:142–5. 13. Reuss ML, Paneth N, Pinto-Martin JA, Lorenz JM, Susser M. The relation of transient hypothyroxinemia in preterm infants to neurologic development at two years of age. N Engl J Med 1996;334:821–7. 14. Paul DA, Leef KH, Stefano JL, Bartoshesky L. Thyroid function in very-low-birth-weight infants with intraventricular hemorrhage. Clin Pediatr (Philadelphia) 2000;39:651–6. 15. Paul DA, Leef KH, Voss B, Stefano JL, Bartoshesky L. Thyroxine and illness severity in very low-birth-weight infants. Thyroid 2001;11:871–5. 16. Leviton A, Paneth N, Reuss ML, Susser M, Allred EN, Dammann O, et al. Hypothyroxinemia of prematurity and the risk of cerebral white matter damage. J Pediatr 1999;134:706–11. 17. van Wassenaer AG, Kok JH, Briet JM, Pijning AM, de Vijlder JJ. Thyroid function in very preterm newborns: possible implications. Thyroid 1999;9:85–91.
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Official Journal of the European Paediatric Neurology Society
Original article
T3 replacement does not prevent excitotoxic cell death but reduces developmental neuronal apoptosis in newborn mice Gergely Sa´rko¨zya,b,1, Elke Griesmaiera, Xiangying Hea,c, Klaus Kapelaria, Martina Urbaneka, Georg Simbrunera, Pierre Gressensd, Matthias Kellera,,1 a
Department of Paediatrics IV, Neonatology, Neuropediatrics and metabolic diseases, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck, Austria b 2nd Department of Paediatrics, Semmelweis University Budapest, Hungary c Department of Pediatrics, The Third Affiliated Hospital of Zhengzhou University, PR China d INSERM U 676 and Service de Neurologie Pe´diatrique, Hoˆpital Robert Debre´ 48, Blvd Serurier, F-75019 Paris, France
art i cle info
ab st rac t
Article history: Received 25 September 2006
Background: Periventricular leukomalacia (PVL) is a major cause of neurological handicap
Received in revised form
in pre-term infants. At present, there are no effective or causal therapies available. Thyroid
26 November 2006
hormones play an essential role in brain development and are reported to be decreased in
Keywords:
Hypothesis: Excitotoxic brain damage of newborn mice decreases thyroid hormone
pre-terms and following brain injury in adults.
Brain injury Newborn Excitotoxicity T3
concentrations. Exogenous T3 administration restores thyroid hormone levels and reduces perinatal brain damage in an animal model of PVL.
Design and method: To create white and gray matter (WM/GM) lesion mimicking several key aspects of PVL, we injected ibotenic acid (Ibo), a glutamate analog, into the right hemisphere (intracranially (i.c.)) of 5-day-old mice. T3 (10 mg/kg body weight (bw)) was injected intraperitoneally (i.p.) 1 h or repeatedly 1/24/48/72/96 h post-insult. We determined lesion size, number of apoptotic cells in WM/GM and serum T3/T4 concentration at 24 and 120 h after injury. Serum T3/T4 concentration was also determined before and 1 and 2 h after T3 administration.
Results: Excitotoxic brain damage did not alter serum T3/T4 concentrations within 120 h of injury. Serum T3 levels were distinctly elevated within 1 h of T3 injection; however, this elevation was relatively short-lived (half-life estimated to be less than 12 h). Neither single nor repetitive T3 treatment regimen reduced excitotoxic lesion size, but it did reduce apoptosis.
Corresponding author. Tel.: +43 512 504 81567; fax: +43 512 504 27766.
E-mail address: [email protected] (M. Keller).
abbreviations: bw, body weight; T3, Triiodthyronine; T4, thyroxine; i.c., intracranial; i.p., intraperitoneal; PBS, phosphatebuffered saline; PVL, periventricular leukomalacia 1 Both authors contributed to the same part. 1090-3798/$ - see front matter & 2006 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ejpn.2006.11.009
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Conclusions: T3 replacement does not prevent excitotoxic cell death, but it does reduce developmental neuronal apoptosis, which could participate to the beneficial neuropsychological effects of hormone therapy. Further study is therefore warranted. & 2006 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
Pre-term infants are at high risk for brain damage and subsequent neurological sequelae. Depending on their birth weight, 20–50% of these infants suffer severe motor disabilities and cognitive deficits.1,2 Aside from the socioeconomic effects and high costs to the health care system, the adverse neurological outcome imposes a severe burden to both the child and parents. Damage to white matter (WM), including periventricular leukomalacia (PVL), is one of the most common brain injury occurring in pre-term infants. Inflammation,3,4 hypoxia/ischemia, free oxygen radical formation5 and excitotoxicity6–8 are all pathogenic mechanisms known to mediate this injury. Additionally, a sudden decline in hormone levels (for instance cortisol, vasoactive intestinale polypeptide and thyroxine) is currently considered to be a risk factor for brain damage and increased mortality in pre-term infants.9,10 Very early pre-term infants are deficient in thyroid hormones because (i) premature infants suddenly lack their mother’s thyroid and (ii) because their hypothalamo–pituitary–thyroid and thyroid metabolism systems have not matured at this developmental stage of life. The result is a period of transient hypothyroxinemia, during which bound and free plasma concentrations of thyroxine (T4) and triiodothyronine (T3) are low.11 In several cohort studies, very low concentrations of T3 or T4 are associated with an increased risk of impaired developmental outcome.11–13 Further, low serum thyroxine, as determined by initial newborn screening, was associated with intraventricular hemorrhage and higher mortality in very low birth weight infants.14,15 After adjusting for potential confounders, such as low gestational age and measures of illness severity, premature infants with hypothyroxinemia had twice the risk of developing sonographic echolucency (indicating PVL) than their peers with higher thyroxine levels.16 It was therefore speculated that the additional T3/T4 substitution might improve neurological and neurodevelopmental outcome in pre-term infants. Several clinical trials have assessed whether exogenous T3/T4 administration improves neurological outcome of pre-terms17,18; however, the results remain controversial.19 Of note, subgroup analysis comparing infants with and without brain damage was not possible in these clinical trials, and thus a protective effect of T3/T4 administration could have been overlooked. Although a matter of particular interest, it is not known whether low T3/T4 values are associated with brain damage (i.e., ‘‘markers’’ of damage), or whether they possess a causal relationship (i.e., ‘‘inducers’’ of damage). In adults, brain damage per se is known to decrease T3 levels20; whether this happens also in newborns has not been investigated.
On this basis, we hypothesized (i) that excitotoxic brain damage decreases total T3/T4 levels in serum; and (ii) that exogenous systemic administration of T3 is neuroprotective against NMDA receptor-mediated brain injury in an animal model of excitotoxic perinatal brain damage mimicking several key aspects of PVL.
2.
Materials and methods
2.1.
Materials
Ibotenic acid (Ibo) tained from Biotrend (Germany), phosphate-buffered saline (PBS) from Invitrogen (Austria) and 3,30 ,5-triiodo-L-thyronine sodium salt from Sigma (Saint Louis, Missouri, USA). Test-kits and reagents: anti-cleaved caspase-3 antibody (Cell Signaling, New England Biolabs, Germany), biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA), streptavidin–biotin complex (Vectastain ABC kit; Vector Laboratories, Burlingame, CA, USA), Immu-Mount (Thermo-Shandon), Diaminobenzidine (Fluka, Buchs, Switzerland).
2.2.
Animal model of excitotoxic brain injury
We used the animal model of excitotoxic brain damage as described previously.21 Briefly 5-day-old CD1 mice were bred at the animal facility of the Medical University of Innsbruck, weighed, and mice with a body weight of 370.3 g at day P5 were included in this study. They were housed under standard conditions in a conventional animal facility with their dams. Adequate measures were taken to minimize pain or discomfort, complying with the European Community guidelines for the use of experimental animals. All pups investigated were anesthetized with isoflurane and kept under a warming lamp. Intracranial (i.c.) injections were performed with a 26-G needle on a 50 ml Hamilton syringe mounted on a calibrated micro-dispenser. The needle was inserted stereotactically 2 mm under the external surface of the scalp in the fronto-parietal area of the right hemisphere, 2 mm from the midline in the lateral–medial plane and 3 mm from the junction between the sagittal and lambdoid sutures in the rostro-caudal plane. Two 1 ml boluses of Ibo (5 mg/ml) were injected 2 and 1 mm under the surface 20 s apart. The needle was left in place for 20 s after the second bolus to avoid leakage. This produces a lesion in WM that mimics the PVL observed in pre-term neonates and striatal/cortical plate injury in full-term human infants. The development of the lesion in this model can be blocked by treatment with NMDA receptor antagonist.21 After injection, the animals were returned to their dams. Histopathology confirmed that the tip of the needle had always reached the periventricular WM.
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2.3.
Experimental groups and study design
Animals from at least four separate litters received either no insult (control) or an i.c. injection of ibotenate (10 mg/2 ml) on post-natal day 5 to create WM and GM lesions similar to those observed in human PVL. The animals of these two groups were further subdivided into five treatment groups: (1) no i.c. or intraperitoneal (i.p.) injections; (2) a control group of one single i.p. injection of vehicle (PBS); (3) a treatment group of a single i.p. injection of T3 (10 mg/kg bodyweight (bw)); (4) a control group of 5 i.p. injections of PBS; and (5) a treatment group of 5 i.p. T3 injections (10 mg/kg bw). Groups 2 and 3 received their PBS or T3 administration 1 h post-injection of ibotenate, while groups 4 and 5 received their PBS or T3 administrations at 1, 24, 48, 72 and 96 h following ibotenate injection. Groups 2 and 3 were analyzed with respect to lesion size, apoptosis, and T3/T4 serum concentrations 24 h following lesion setting, while groups 4 and 5 were analyzed after 120 h. The absolute dose of T3 was adapted daily to the current body weight.
2.4.
Lesion size determination
Mouse pups were euthanized by decapitation either 24 or 120 h following the excitotoxic injury. Brains were immediately immersed in 4% formaldehyde and fixed for 72 h. Following paraffin embedding, blocks were sliced into 10 mm coronal sections. Every third section was stained with cresyl violet and the lesion size was determined as described elsewhere.21,22 Briefly, neocortical and WM lesions can be defined by the maximal length of three orthogonal axes: a horizontal and radial axis in the coronal plane and one in the fronto-occipital axis (in a sagittal plane). In previous studies, we have shown an excellent correlation between the maximum size of the different diameters and excitotoxic lesions. Based on these observations, we serially sectioned the entire brain in the coronal plane. This permitted an accurate and reproducible determination of the maximum sagittal frontooccipital diameter (which is equal to the number of sections where the lesion was present multiplied by 10 mm) and this was used as an index of the volume of the lesion. Two observers independently determined lesion size, each blinded to the treatment groups being analyzed. Data are presented as mean length of the lesion in the rostro-caudal axis7SEM.
2.5.
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streptavidin–biotin complex (Vectastain ABC kit; Vector Laboratories, Burlingame, CA, USA). Visualization was performed using diaminobenzidine as the chromogen. Following multiple rinses in distilled water, sections were dehydrated, cleared in xylene and coverslipped. Sections from similar anatomical areas were processed simultaneously. Labeled cells in the neocortical layers and underlying WM at the level of ibotenate-induced lesions (edges and maximum of the lesion) in the injured and noninjured hemispheres, as well as in the basal ganglia were counted by one observer, who was blinded to the treatment group. Six brains from each group were randomly selected, and 8–12 brain sections of 10 mm thickness were examined from each brain. Data are given as mean number of positive cells in WM and GM per brain7SEM.
2.6.
Determination of T3 and T4 in serum
To assess the effect of brain damage, and of single and repetitive T3 administration on total T3 and T4 serum concentration in newborn mice, we determined T3 and T4 serum concentration in (1) healthy mice with no intervention; (2) brain injured mice (i.c. injection of ibotenate)+PBS i.p.; (3) brain injured mice (i.c. injection of ibotenate)+one single T3 i.p. injection; and (4) brain injured mice (i.c. injection of ibotenate)+repetitive T3 i.p. injections. Measurement of serum T3/T4 concentrations was completed on days P5, P6 and P10 for group 1; days P6 and P10 for group 2; day P6 for group 3 and day P10 for group 4. Determination of total serum T3/T4 from blood samples was performed using an electrochemiluminescence immunoassay (ELICA) for quantification (Roche, Mannheim, Germany) according to the manufacturer’s instructions.
2.7.
Statistics
Two standard statistical tests were employed in this study. An unpaired Student’s t-test was employed when only two experimental groups were compared. For multiple comparisons, and ANOVA was first employed to determine if any of the treatment groups statistically differed, followed by t-tests with Dunnet’s correction in order to determine which groups differed. Statistical differences were considered to be significant at a level of po0.05.
Immunohistochemistry for cleaved caspase-3
3. Sections (10 mm) of formaldehyde-fixed and paraffin-embedded mouse brain were deparaffinized in xylene and passed through graded alcohols. Endogenous peroxidase activity was quenched by incubation in 2% hydrogen peroxide in methanol for 30 min. Heat-induced antigen retrieval was performed using 10 mM citrate buffer (pH 6.0), for 15 min in a microwave oven. After blocking with 10% fetal calf serum (in PBS), sections were exposed to a rabbit anti-cleaved caspase-3 (Asp 175) antibody (1:500; Cell Signaling Technology via New England Biolabs GmbH, Frankfurt, Germany). The sections were subsequently incubated for 45 min at 25 1C with biotinylated goat anti-rabbit IgG (1:100; Vector Laboratories, Burlingame, CA, USA) and then for 45 min at 25 1C with a
Results
3.1. Excitotoxic brain damage does not decrease total T3/ T4 serum concentrations in newborn mice As displayed in Fig. 1, excitotoxic brain damage is not associated with a decrease in serum T3 and T4 concentrations (n ¼ 3–7), at least within 120 h of the initial insult. In control (untreated) animals, we observed that T3 levels significantly decreased (Fig. 1a) and T4 levels significantly increased (Fig. 1b) between days P6 and P10. The serum T3/T4 levels in the two excitotoxic injury treatment groups (Ibo+PBS/Ibo+T3) were not different when compared to their time-matched controls.
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a
24 h post-insult (P6) and 120 h post-insult (P10). In the case of the latter, single and repetitive T3 injection treatments were compared. There were no significant differences in lesion size when the treatment groups were compared to their timematched control. Although there were tendencies for the lesion sizes to change between day P6 and P10 (smaller for WM; larger for GM), these differences were not found to be significant.
+ single/5 x PBS i.p.
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3.3. Exogenous T3 decreases developmental apoptosis in the newborn brain
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6
4.
Discussion
4
2
n=3
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Fig. 1 – Quantitative effects of age, excitotoxic brain damage and T3 supplementation on total T3 (a) and T4 (b) concentrations in serum. Animals were treated with a single dose (P6) or with 5 repetitive doses (beginning on P6, every 24 h until P10) of T3 or PBS respectively. Excitotoxic lesions were produced on P5 and serum T3 and T4 concentrations were analyzed on days P5, P6 and P10. Bars represent mean concentration of T3 and T47SEM. Numbers in the bars represent the number of animals used in each experimental group (n ¼ 3–7).
Notably, a single injection of T3 (10 mg/kg bw), administered 1 h post-excitotoxic insult, did not cause alterations in T3/T4 levels measured at 24 (P6) and 120 (P10) h post-insult. The T3 injections, therefore, result in only a transient elevation in T3 levels, which were observed within 1–2 h of the T3 injection (pre-T3 injection: 145712 ng/dl; 1 h post-T3 injection: 1410790 ng/dl, po0.01 compared to pre-injection; 2 h postT3 injection: 1356798 ng/dl, po0.01 compared to pre-injection).
3.2.
As shown in Fig. 3, repetitive T3 treatment significantly reduced apoptosis. Excitoxicity per se slightly increased the number of cells positive for caspase-3 cleavage (a marker of apoptosis) at 120 h after the insult in GM and WM compared to the undamaged hemisphere. In both, the damaged (Fig. 3 right), but also in the undamaged (Fig. 3 left) hemispheres the number of cells positive for caspase-3 cleavage was significantly reduced in animals treated with T3.
I.p. T3 does not reduce excitotoxic brain injury
Fig. 2 shows that following excitotoxic insult, injection of T3 (10 mg/kg bw) does not reduce WM (Fig. 2a and b) or GM (Fig. 2a and c) lesions. Lesion size was assessed at two time points:
In the present study, we show that exogenous T3 administration does not decrease NMDA receptor-mediated excitotoxic brain damage in newborn mice. In contrast to our hypothesis, neither a single nor repetitive i.p. administrations of T3 reduced WM or GM lesion size. Furthermore, excitotoxic brain injury in newborn mice per se did not alter total T3 and T4 serum concentrations within 120 h of the insult. We systematically assessed the age-dependent physiological changes in T3 and T4 concentrations and found that both hormone levels changed between days P6 and P10. This highlights the need for similar studies to have the proper time-matched controls. Of note, exogenous T3 administration did not result in sustained changes in T3 or T4 levels (Fig. 1). The half-life of injected T3 is known to be relatively long in newborn humans (24 h), but it can be as short as 6 h in other newborn mammals, such as piglets.23 The half-life of T3 in mice is not known, but our data suggests that it is less than 12 h. The primary aim of the present study was to determine if exogenous T3 administration could reduce excitotoxic brain injury. The T3 dosage employed was within the range of doses used in other studies,24,25 and was sufficiently low enough that it did not cause hyperthyroidism (50 mg/kg bw26; i.p. injection of T3 resulted in a marked increase in T3 serums levels (within 1 h); previous studies indicate that T3 levels in venous blood reach almost all brain centers (including GM, cerebellum, caudate/putamen, hippocampal pyramidal layer, dentate gyrus, thalamic nuclei, superior and inferior colliculi, brain stem nuclei and ventricles) within 3 h.27 Injection of T3 did not ameliorate ibotenate-mediated excitotoxic cell death. However, since injected T3 appears to have a relatively short half-life in the bloodstream (less than 12 h), future studies should attempt a procedure that results in sustained T3 elevation. Administration of T4, for example,
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Fig. 2 – (a) Representative photographs (5 magnification) of brain slices stained with cresyl violet. The photographs display the cyst and cortical atrophy in the PBS- and T3-treated mice following excitotoxic injury. (b/c) Quantitative effect of T3 i.p. treatment on ibotenate-induced white (b) and gray matter (c) lesions. Lesions were produced on P5 and analyzed on days P6 and P10. Bars represent mean length of the lesions in the fronto-occipital axis 7SEM. Numbers in the bars represent the number of animals used in each experimental group (n ¼ 8–12).
may result in higher intracellular levels of T3 and may have a longer half-life. Such protocols, however, were beyond the scope of the present study. Injection of T3 was found to reduce developmental neuronal apoptosis. Neuronal apoptosis is a normal physiological process of brain development, the functional significance of which remains unclear (see Ref.28 for a review). The consequences of reducing (or delaying) the developmental apoptosis of neocortical neurons are unknown; whether it is beneficial or detrimental likely depends on physiological or patho-physiological state of the brain at the time of intervention. Behavioral studies will be necessary to determine the
functional correlates of this histological finding. However, there is a potential for T3 administration to be beneficial in pre-term infants at risk of neuronal loss. This is consistent with other clinical data indicating better schooling and motor outcomes in children who were supplemented with T4 following very early (o27–28 weeks) premature birth.29 On a cautionary note, however, the reverse was true when T4 was administered to slightly older infants of 29 weeks’ gestation.29 In our experimental setting, T3 supplementation did not alter the T4 serum concentration in newborn mice. However, we cannot exclude the possibility that T3 supplementation may result in a significant reduction in the serum T4 level of
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Fig. 3 – (a) Representative photographs, 20 magnification) of brain slices immuno-stained for activated caspase-3 (a marker of apoptosis) in PBS- and T3- and treated mice indicating the decrease in apoptosis in undamaged brain tissue by T3 treatment. (b) Quantitative effects of T3 (10 lg/kg) i.p. treatment on ibotenate-induced apoptosis in gray matter and the underlying white matter. Data are presented as number of cleaved caspase-3 positive cells 7SEM in the right (damaged) and left (undamaged) hemisphere at day P10. Asterisks indicate statistically significant differences from PBS-treated controls (po0.05, Student’s t-test). Numbers in the bars represent the number of animals used in each experimental group.
human pre-term infants, thereby resulting in a critical reduction in brain T3. In this context, T3 supplementation is not recommended until its safety is confirmed. In conclusion, exogenous T3 administration does not reduce excitotoxic cell death. T3 administration appears to reduce developmental neuronal apoptosis, which could participate to the beneficial neuropsychological effects of hormone therapy in pre-term infants less than 28 weeks of gestation.
Acknowledgment We thank Darcy Lidington for carefully proof reading this manuscript and his valuable contributions.
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