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Different types of pharmacologic treatment have shown neuroprotection against HI in immature animals(1–6). In most of these studies neuropathology was used as the sole outcome at 1-14 d after HI. The injury occurs, however, in different phases during ischemia, and matures during hours to several weeks after the insult(7–9) implicating the need for introduction of novel criteria of outcome.

In the core of the infarct, all cell types die during or directly after HI. In the penumbral zone the tissue remains viable at least 0-6 h after ischemia(10). Areas subjected to milder degrees of HI may undergo selective neuronal death (necrosis or apoptosis) with a time-course that is further delayed(11, 12). Cortical lesions will lead to secondary damage and atrophy of subcortical regions, particularly in the thalamus(13, 14).

During development, hypoxia may also affect neurite out-growth and synapsogenesis with subsequent long-term impairments of cognitive functions(15). Tissue repair and compensatory mechanisms may further modulate the extent of primary and secondary lesions and their functional consequences, especially in the immature brain(16–18). If this complex scenario is considered, the volume of the infarct a few days after HI cannot be anticipated to offer satisfactory information with regard to structural and functional outcome.

The behavioral consequences of perinatal asphyxia are of great importance, because HI encephalopathy in children often causes functional and intellectual deficits(19, 20), and to reveal the true functional disability it is important to do long-term follow-up studies. Likewise, to study the effects of drug intervention in experimental HI, it seems justifiable to consider at least three different end points: the acute extent of brain injury, the long-term deficit of brain tissue, and the functional consequences of the brain lesion after most compensatory and reparative phases have been passed. The validity of such a concept is supported by e.g. hypothermia reduces hippocampal cell damage in the short-term but not in the long-term perspective(21). Further experience from adult animals has shown that environmental factors can improve the functional outcome without affecting the lesion size(22, 23). Another study provides evidence for reduction of the lesion size without any functional protection(24). These studies indicate that evaluation of long-term functional outcome introduces a complementary cerebroprotective dimension. Environmental influences(22) and pharmacologic therapy(15) may improve functional outcome through mechanisms unrelated to the salvage of the brain tissue. In fact, intervention during the phase of functional adaptation may well prove to be very important in the immature brain with its greater plasticity and compensatory capacity(18, 25, 26).

Therefore, the model of neonatal HI in 7-d-old rats according to Rice et al.(27) was applied. This model produces brain injury preferentially in the territory of the middle cerebral artery, including sensorimotor cortex, thalamus, and striatum, which are all critical for maintenance of sensorimotor function in rats(28–32). Based on experience of cerebral lesions in adult(23, 33–35) or neonatal(17) rats, four tests were selected and refined for evaluation of sensorimotor deficits. These results were related to the total hemispheric loss of brain tissue with normal histology evaluated with H&E staining and MAP-2 immunoreactivity.

METHODS

Protocol and operative procedures. Wistar rat pups of both sexes were used. At postnatal d 7, 11 pups were exposed to HI as follows. The pups were anesthetized with halothane (3.0-3.5% for induction and 1.0-1.5% for maintenance) in a mixture of nitrous oxide and oxygen (1:1). The left common carotid artery was dissected and cut between double ligatures of prolene sutures (6-0). The duration of anesthesia was <10 min. After the surgical procedure, the wounds were infiltrated with a local anesthetic. The pups were left to recover for at least 1 h. The litters were then placed in a chamber perfused with a humidified gas mixture (7.70 ± 0.01% oxygen in nitrogen) for 100 min. The temperature in the gas chamber was kept at 36 °C. After hypoxic exposure the pups were returned to their dams. They were reared at 20 °C environmental temperature with a light:dark cycle of 12:12 h and food and water ad libitum. At postnatal d 28 the pups were weaned and separated by sex. Three to five animals were put into individual boxes. There were three different control groups: sham operated without subsequent hypoxia (the artery was just visualized, n = 5), ligated animals without subsequent hypoxia (n = 4), and total controls without both hypoxia and ischemia (n = 11). All animal experiments were approved by the ethical committee of Göteborg (no. 1-95).

Sensorimotor tests. The rats were tested at postnatal age 42 d. The last days before testing the rats were handled by the examiner. The tests were carried out during the first hours of the light portion of the 12/12 h light:dark cycle. Each rat participated 15-20 min per trial. All tests were performed in the same day and in the same order for all rats.

In the modified grip-traction test(34) the muscle strength of the rat was tested by its ability to hang on to a horizontal rope (0.6-cm diameter plastic tube was placed horizontally 45 cm above the table) by its forepaws (Fig. 1A). Time to falling (maximum 60 s) and ability to bring up the right hindlimb onto the rope (contralateral to the ligated side) were noted.

Figure 1
figure 1

(A-D) These line drawings illustrate the different functional tests. (A) The rat's ability to hang on to a horizontal rope is noted in the grip-traction test. (B) The number of misplaced fore- or hindlimb movements where the paws fall through the grid bars is registered in the foot-fault test. In the illustration the rat makes a right-sided foot-fault with the hindlimb. (C) In the postural reflex test, the rat is held above a table, and extension or flexion in the right forelimb is noted. This rat has a flexed right forepaw and is scored 1. (D) Limb placing after different sensory stimuli is scored in the limb-placing test. Both illustrations demonstrate a right-sided score 2 and a left-sided score 0.

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In the foot-fault test(17) the rat was placed on a horizontal grid floor (50 × 40 cm, square size 3 × 3 cm, wire diameter 0.4 cm) (Fig. 1B). The foot-fault was defined as when the animal misplaced a fore- or hindlimb and the paw fell through between the grid bars. Number of foot-faults was noted during 2 min. Only the side difference of foot-faults was used for the statistical evaluation to eliminate the influence of the extent of activity in different rats.

In the postural reflex test(33), the rat was held by the tail 50 cm above a table (Fig. 1C). Normal rats extend both forelimbs toward the table (score 0). Rats with brain damage flex the forelimb contralateral to the damaged hemisphere (score 1). Thereafter, the rat was put onto the table, and a lateral pressure was applied behind the shoulder of the rat until the forelimbs slid. This was repeated several times, and a reduced resistance to lateral force toward the right side (contralateral to the brain damage) was considered abnormal (score 2).

In the limb-placing test(23, 35), the rat was held by the examiner, and the fore- and hindlimb placement after different sensory stimuli (see 1-6 below) was noted as: score 0, immediate and correct paw placing; score 1, delayed and/or incomplete correction; score 2, no placing (Fig. 1D). Side differences were noted for each rat. 1) Visual limb placing was tested by lowering the rat toward a table. Normal rats stretch and place both forepaws on the table. 2) Forelimb sensory input was tested with the rat's forelimbs touching a table edge. The cephalic contact was eliminated by lifting up the head of the rat and the contralateral forelimb-placing deficit was revealed. 3) Forelimb placement was tested when the rat was facing the edge of the table. A normal rat placed both forepaws on the table top, whereas the rat with the brain lesion failed to correctly place the contralateral paw when vibrissae contact was lost. 4) Both fore- and hindlimb placement was tested when the rat was held by the examiner and slowly moved laterally toward the edge of the table. 5) The rat was placed on the table and gently pushed laterally toward the edge of the table. A control rat tends to grip onto the edge, whereas an injured rat may drop the fore- or hindlimb contralateral to the injured hemisphere. 6) As 5 above, but the rat was pushed from behind.

Brain sections. The rats were decapitated on postnatal d 48, and the brains were dissected and frozen in dry ice-chilled dimethylbutane. The brains were stored at -80 °C until use. Consecutive coronal cryostat sections for H&E staining and immunohistochemical staining for MAP-2 was done at 10 levels with a 1-mm distance (3.8 mm anterior to 5.2 mm posterior to the bregma). The 10-μm thick sections were thaw-mounted on Polysine™ (Menzel-Gläser, Braunscheveig, Germany) coated slides.

Immunohistochemical staining. The dendrites and soma of the neurons were immunohistochemically stained with a mouse MAb against rat MAP-2 (clone no. HM-2, Sigma Chemical Co., St. Louis, MO). The sections were air-dried for at least 30 min and circled by a Pap-pen (Daido Sangyo, Tokyo) to stop the antibodies from floating out. Sections were fixed for 10 min with 4% paraformaldehyde in PBS (pH 7.4, 0.08 M). After rinsing in PBS, nonspecific binding was blocked for 30 min with 4% horse sera in PBS. The sera was shaken off, and the anti-MAP-2 antibody (dilution 1:2000) was incubated for 60 min. After rinsing twice in PBS, the sections were incubated with a biotinylated horse anti-mouse antibody for 60 min followed by 5 min of treatment with 0.6% H2O2 in methanol. The sections were incubated with avidin-biotin-peroxidase complex for 60 min (Vector laboratories, Burlingame, CA). Then the glasses were placed in sodium acetate buffer (pH 6.0, 0.1 M) for 10 min. Finally the immunoreactivity was visualized with 50 mg of 3,3-diaminobenzidine enhanced with 1.5 g of ammonium nickel sulfate, 200 mg of β-D-glucose, 40 mg of ammonium chloride, and 1 mg of β-glucose oxidase (Sigma) dissolved in 100 mL of sodium acetate buffer (pH 6.0, 0.1 M). Negative controls were performed using the same procedure described above in the absence of primary antibody. These sections were devoid of immunoreactivity.

Volume measurement. The measurement of infarct volume in MAP-2 and H&E sections was performed with an image-analyzing system which consisted of a Nikon Optiphot-2 microscope, a videocamera (CCD72, Dage-MTI, Michigan City, IN), a digitizing unit attached to the videocamera (CCD72, Dage-MTI), and a Macintosh Quadra 900 computer equipped with a videocard. The software program Voltum for LabVIEW Run-Time 2.2.1 (National Instruments, Austin, TX) was used to measure the area of damaged and undamaged tissue in the left and right hemisphere and calculating the volume of brain damage (expressed as 100% - volume of intact left hemisphere as percent of the right hemisphere).

Statistics. Chi-square analysis was used for tests based on the scoring system. Mann-Witney U test was used for differences in body weight, time to falling in the grip-traction test, and foot-fault test.

RESULTS

There was no mortality in either the HI or the control group, and there was no difference in sex distribution between the groups. The body weight at postnatal d 7 was 13.7 ± 1.07 g (mean ± SD) with no difference between the groups. Mean body weight at postnatal d 42 was 139 ± 18.5 g in the HI group and 158 ± 29.2 g in the control group (NS).

All rats exposed to HI were lesioned in the left hemisphere, whereas all control rats were uninjured. The volume of intact left hemisphere was not different from the right hemisphere in the different control groups (Table 1). The mean percent volume deficit of the left hemisphere compared with the right based on H&E-stained sections and MAP-2 sections is shown in Table 1. There was a close correlation in mean percent volume deficit between H&E and MAP-2 sections (Spearman's rank correlation p < 0.0001; r = 0.999).

Table 1 The mean percent volume deficit of the left hemisphere compared to the right based on H&E and MAP-2 stained sections in the HI and the different control groups (mean ± SD).
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In the grip-traction test, the control rats hung on to the rope 39.6 ± 21.9 s compared with 18.0 ± 18.4 s (mean ± SD) in the HI group, p < 0.01. In the control group, 90% of the rats were able to bring up their right hindlimb compared with 27% in the HI group, p < 0.001. In the foot-fault test, limb misplacements in control animals were symmetrical, i.e. tended to occur equally often in either left or right side. The difference (number of foot-faults right-left side) was -0.2 ± 0.9 in the control group and 4.3 ± 3.5 in the HI group (mean ± SD), p < 0.0001. In the postural reflex test 95% of the control rats had score 0, and none of them had score 2. In the HI group, 73% of the rats had score 1 or 2, i.e. there was an obvious difference in frequency distribution, p < 0.001 (Fig. 2). In the limb-placing test, all the control rats behaved symmetrically, whereas in the HI group the median side difference (right-left) was 6, p < 0.001 (Fig. 3). Functional outcome was plotted against loss of ipsilateral brain tissue volume (Fig. 4). The total rank was obtained by pooling the ranks of the separate tests. There was a correlation between functional and neuropathologic outcome when all groups were included (p < 0.001; r = 0.842). There was, however, no significant correlation between functional and neuropathologic outcome within the HI group (p = 0.60; r = 0.20). There was no difference between female and male rats in the size of the brain damage, in the separate test results, and in the total rank. In spite of a slight overlap between results in HI and control groups, all the HI animals diverged from normal values in at least one test (Fig. 5).

Figure 2
figure 2

Score distribution in the postural reflex test. All control groups were pooled into one group. Score 0, extension of both forelimbs; score 1, flexion of the right forelimb; score 2, reduced resistance to lateral push. The difference in distribution between the groups was significant (X2 analysis, p < 0.001).

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Figure 3
figure 3

In the limb-placing test, the side difference of right-left fore- and hindlimb placement after different sensory stimuli was noted for each rat. All control rats were pooled into one group. The distribution of right-left limb-placing score differed significantly between the groups (X2 analysis, p < 0.001).

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Figure 4
figure 4

Total rank of the four tests (all tests were ranked separately and then pooled together) was plotted against the loss of ipsilateral brain tissue volume. There was a correlation between functional and neuropathologic outcome (p < 0.001; r = 0.842).

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Figure 5
figure 5

All the HI animals diverged from normal values in at least one test, whereas 95% of the control rats behaved normally in three of the four test. Normal values in grip time >20 s, foot-fault right-left side = 0, postural reflex test and limb-placing score difference = 0. The difference in distribution was statistically significant (X2 analysis, p < 0.0001) between the groups.

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DISCUSSION

In this study, four sensorimotor tests were evaluated in the rat model of neonatal HI brain damage and related to the loss of brain tissue. All HI animals had sensorimotor deficits or asymmetries in at least one test. There was, however, some overlap with control animals in most tests, which emphasizes the need for more than one test to increase the sensitivity and specificity of the test protocol. In all the separate tests, the HI rats performed significantly worse than did the controls. The majority (95%) of the control rats were completely normal in three of the four tests. Brain injury was morphologically evaluated 6 wk after HI, and the loss of H&E staining tissue correlated closely with the loss of MAP-2 immunostaining in this model at this late time point (Table 1).

We found a moderate correlation between the size of damage and total functional rank if all animals were included, but not if evaluated within the group with brain injury (Fig. 4). One explanation for the poor correspondence within the HI group is that all the HI rats had extensive brain damage (Fig. 4) that decreased the chance in succeeding with a correlative analysis. Another suggestion to poor correlation between structure and function is an asymmetrical organization of brain functions(36), but there is no general agreement on this topic, and opposing results have been reported(37). Yet another cause might be that, even if the tests were chosen specifically for sensorimotor cortex lesions, they correlate better to different regional tissue loss than to the whole hemispheric lesion. Nevertheless, the abilities were generally better than expected in the different test paradigms, considering the large lesions in these rats, which may relate to the great adaptive capacity of the immature brain. For example, studies with transplantation of neocortical tissue in cortical lesions have shown a greater network of connections between the graft and the host in the immature brain(38) compared with the adult brain(39, 40). Furthermore, the thalamic atrophy secondary to cortical lesions can be attenuated(41, 42) and functional deficits can be improved(43) in neonatal rats.

The spontaneous motor activity was normal in the lesioned rats, which could partly be an effect of aberrant corticospinal projections from the undamaged (contralateral) hemisphere that do not cross the midline. These fibers are spared in this situation with an ipsilateral lesion and masks the damage until the contralateral cortex is lesioned in the same way, as shown by Barth and Stanfield(17) 42 d after cortical ablation in neonatal rats.

Some of the HI rats had a mild left-sided ptosis most probably as a result of touching the pericarotid sympathetic nerve fibers during the operation(44). To exclude the possibility that these rats performed less well than did total controls because of affected vision we included some sham-operated rats. However, there was no significant difference between the three control groups (sham, ligated, and not ligated controls), suggesting that ptosis is not a serious artifact in functional evaluation.

Earlier behavioral observations done in this model are limited and do not include sensorimotor functions in the long-term perspective(27, 45–47). They found defective motor skills soon after the damage (10 min to 9 d after HI). The tests performed at postnatal d 30-37 were focused at cognitive functions (Morris water maze, T maze, and the shock avoidance test) related mostly to the hippocampus formation, but the most constantly affected region in both mild and severe lesions in this model is the cerebral cortex(27, 44, 48).

In conclusion, this study describes sensorimotor tests that could be used for long-term evaluation of functional deficits after HI in rats. The data support the concept that lesion size does not correlate strictly with functional outcome(24, 37, 49) and that assessment of the acute and the long-term extent of the lesion and functional outcome are complementary and not replaceable modalities for evaluation of outcome after HI. We plan to test this combined approach for functional and morphologic long-term evaluation in studies on neuroprotective interventions.