Effects of IL-3 on CA1 Neuronal Density, Volume of the CA1 Pyramidal Cell Layer, and Response Latency In Vivo.
We first investigated CA1 neuronal density, volume of the CA1 pyramidal cell layer, and response latency in sham- operated and ischemic gerbils with vehicle infusion to ascertain that they were significantly reduced by a 3-min ischemic insult. The mean CA1 neuronal density and volume of the pyramidal cell layer along 1.5 mm linear length of the hippocampal CA1 field in sham-operated animals were 248.7 ± 12.4 cells/mm and 0.390 ± 0.02 mm3/per section, respectively, and those of 3-min ischemic gerbils infused with vehicle alone were 127.3 ± 31.5 cells/mm and 0.268 ± 0.03 mm3/per section, respectively. The mean response latency in sham-operated animals was 230.8 ± 40.5 s, and that in vehicle-infused ischemic gerbils was 124.5 ± 28.8 s. There were significant differences in CA1 neuronal density (U = 0, P <0.01), volume of the CA1 pyramidal cell layer (U = 0, P <0.01), and response latency (U = 0, P <0.01) between the two groups (Fig. , A–C). In histological sections, the CA1 region of ischemic gerbils exhibited a marked decline in viable neurons compared with the CA1 field of sham-operated animals (Fig. , D and E).
Figure 1 (A and B) Effects of intracerebroventricular IL-3 infusion on CA1 neuronal density (A) and volume of the CA1 pyramidal cell layer (B) in ischemic gerbils. The infusion of IL-3 started 2 h before or just after 3-min ischemia, and continued for 7 d. Significant (more ...)
We next investigated whether IL-3 treatment prevented the ischemia-induced decreases in CA1 neuronal density and volume of the CA1 pyramidal cell layer. Hippocampal CA1 neurons in ischemic gerbils infused with IL-3, starting 2 h before ischemia, outnumbered significantly those in ischemic gerbils infused with vehicle (64 or 320 ng/d of IL-3 versus vehicle in ischemic gerbils: U = 20, P <0.05, or U = 6, P <0.01, respectively [Fig. , A, F, and G]). The volume of the CA1 pyramidal cell layer per section was also significantly larger in the IL-3–treated than in vehicle-treated ischemic gerbils (64 or 320 ng/d of IL-3 versus vehicle in ischemic gerbils: U = 12, P <0.05, or U = 1, P <0.01, respectively [Fig. B]).
The prevention by IL-3 infusion of ischemic neuronal damage in the hippocampal CA1 field was further reinforced by the results of passive avoidance tests. The infusion of IL-3, starting 2 h before ischemia, caused a significant dose-dependent prolongation in response latency in the step-down passive avoidance task (64 or 320 ng/d of IL-3 versus vehicle in ischemic gerbils: U = 20, P <0.05, or U = 4, P <0.01, respectively [Fig. C]). The results of passive avoidance experiments correlated well with the neuronal density of the hippocampal CA1 region in sham-operated and ischemic gerbils infused with vehicle or IL-3 (r = 0.770, P <0.05 [Fig. H]).
The effect of postischemic treatment with IL-3 on delayed neuronal death was also investigated. IL-3 infusion in a dose of 64 or 320 ng/d for 7 d, starting just after 3-min ischemia, also prevented the ischemia-induced decreases in the number of CA1 neurons and volume of CA1 field (64 or 320 ng/d of IL-3 versus vehicle in ischemic gerbils: U = 10, P <0.05, U = 8.5, P <0.05; or U = 7, P <0.01, U = 0.5, P <0.01, respectively) and caused a significant prolongation in response latency compared with that of vehicle-treated ischemic gerbils (64 or 320 ng/d of IL-3 versus vehicle in ischemic gerbils: U = 10, P <0.05, or U = 5, P <0.01, respectively [Fig. , A–C]).
Effects of IL-3 on the Number of Synapses and Fine Structures of Pyramidal Neurons in the Hippocampal CA1 Field.
In line with the results of the light microscopic observations and passive avoidance task, electron microscopy showed that intact synapses within the stratum moleculare, stratum radiatum, and stratum oriens of the hippocampal CA1 region were more numerous in IL-3– than in vehicle-treated ischemic gerbils (64 ng/d of IL-3 versus vehicle in the stratum radiatum of ischemic animals: U = 12, P <0.05; 320 ng/d of IL-3 versus vehicle in the individual strata of ischemic animals: U = 7, P <0.05; U = 8, P <0.05; U = 5, P <0.01 [Fig. A]). Under light microscopy, vehicle-treated hippocampal CA1 neurons surviving 7 d after 3-min ischemia seemed to be intact (Fig. E). However, a careful observation of the neurons with an electron microscope revealed that significant numbers of neurons were in the course of nuclear chromatin fragmentation and/or condensation to different degrees (Fig. , B and C); the nuclei of vehicle-treated ischemic neurons at early stages of degeneration had an irregular euchromatin with low electron density compared with intact nuclear euchromatin (Fig. B). On the other hand, most of the surviving pyramidal neurons in the CA1 field of IL-3–treated gerbils retained normal morphological features even 7 d after ischemia (Fig. D). These findings suggest that IL-3 facilitates the survival of hippocampal neurons loaded with ischemic insult in vivo.
Figure 2 (A) Effect of IL-3 on the number of intact synapses in the three strata of the hippocampal CA1 region. The infusion of IL-3 or vehicle was started 2 h before 3-min ischemia, and continued for 7 d. Intact synapses in the strata radiatum (sr), moleculare (more ...) Effects of IL-3 on the Number of TUNEL-positive Neurons in the Hippocampal CA1 Field.
TUNEL staining revealed that many TUNEL-positive neurons were present in the hippocampal CA1 field of 3-min ischemic gerbils with vehicle infusion 7 d after ischemic insult (Fig. A), suggesting that irreversible neuronal degeneration was in progress at this period as deduced from the electron microscopic findings (Fig. , B and C). The 7-d infusion of IL-3 not only prevented delayed neuronal death in the hippocampal CA1 field 7 d after ischemia, but also reduced the number of TUNEL-positive neurons which were in the course of a more delayed degeneration (Fig. , B and C). The count of TUNEL-positive cells in the vehicle-infused ischemic gerbils indicated that without IL-3 treatment, nearly one half of the CA1 neurons surviving 7 d after ischemia undergo a further degeneration within a few days (Figs. A and D). IL-3 treatment precluded the late onset of ischemia-induced neuronal degeneration in a dose-dependent manner (64 or 320 ng/d of IL-3 versus vehicle: U = 4, P <0.05, or U = 0, P <0.01, respectively [Fig. D]).
Figure 3 (A–C) Photomicrographs of TUNEL-positive neurons in the hippocampal CA1 field of ischemic gerbils after 7-d infusion of vehicle or IL-3: vehicle (A); 64 ng/d of IL-3 (B); 320 ng/d of IL-3 (C). The infusion was started 2 h before 3-min ischemia, (more ...) Demonstration of IL-3Rα in the Hippocampal CA1 Field.
Although we assumed that IL-3 infused into the left lateral ventricle reached the hippocampus to rescue ischemic CA1 neurons through its binding to the local receptors, immunohistochemical analysis using the IL-3Rα antibody showed only scattered positive staining in the hippocampal CA1 field of sham-operated gerbils (Fig. A), despite an intense staining in the hippocampal CA3 field, which is known as a site tolerant to ischemia (Fig. B). Expecting that IL-3Rα might be abundantly expressed in the hippocampal CA1 field of ischemic but not sham-operated gerbils with vehicle infusion, we investigated the temporal profile of IL-3Rα expression in the CA1 field during 1–7 d after 3-min ischemia. Occlusion of the common carotid arteries induced a significant increase in IL-3Rα immunoreactive neurons at 2 and 4 d after ischemia (Fig. , C and D). The enhanced immunoreactivity of IL-3Rα began to decline 7 d after ischemia (Fig. E). The IL-3Rα immunoreactions in the hippocampal CA1 field of ischemic gerbils were completely abolished by adsorbing the primary antibody with the homologous antigen (Fig. F). Immunoblot analysis showed a weak but distinct constitutive expression of IL-3Rα with a molecular mass of 70 kD in the hippocampal CA1 field of sham-operated gerbils (Fig. G). The receptor expression in the field increased at 2 and 4 d after ischemia (Fig. G). Thus, the transient upregulation of IL-3Rα expression in a population of hippocampal CA1 neurons after ischemic insult may have made it easy for cerebroventricularly infused IL-3 to act on the neurons. The infusion of IL-3 in ischemic gerbils did not affect IL-3Rα immunoreactivity in the CA1 field (Fig. H), except that 320 ng/d of IL-3 infusion caused a decline in IL-3Rα–immunoreactive CA1 neurons at 4 d after ischemia (Fig. I) compared with immunoreactive CA1 neurons in vehicle-treated ischemic animals (Fig. D). This finding may reflect downregulation of IL-3R in the CA1 field of ischemic gerbils infused with the ligand.
Figure 4 (A–F) Photomicrographs of IL-3Rα–immunoreactive neurons in the dorsal hippocampus: CA1 field of a sham-operated animal with vehicle infusion (A); CA3 field of a sham-operated animal with vehicle infusion (B); CA1 field of vehicle-treated (more ...) Expression of Bcl-xL in the Hippocampal CA1 Field.
If binding to the receptor upregulated transiently after ischemia, centrally infused IL-3 should transmit signals in favor of neuronal survival, leading to the generation of neuroprotective agents in the CA1 neurons. Among Bcl-2 family proteins, Bcl-xL
protein, which suppresses apoptosis, is known to be expressed in the mature central nervous system, and Bcl-xS
protein, which facilitates apoptosis, is barely detectable in the adult brain (21
). Based on the finding that IL-3 precludes apoptotic death of an erythroleukemic cell line by inducing Bcl-2 protein (15
), we speculated that Bcl-xL
might be a candidate for the neuroprotective agents induced by IL-3 treatment.
In sham-operated gerbils, Bcl-xL mRNA was weakly and evenly expressed in pyramidal neurons of the CA1-4 fields and in dentate granule cells (Fig. A). Forebrain ischemia of 3-min duration caused a selective increase in Bcl-xL mRNA expression in the hippocampal CA1 field of gerbils at 1, 2, and 4 d after ischemic insult (Fig. , B and C). Quantitative analysis showed that relative amount of Bcl-xL mRNA increased significantly in the CA1 field at 1, 2, and 4 d after ischemia, and thereafter declined to the control level (Fig. D). No significant changes were observed in the other regions of the hippocampus. The expressions of Bcl-2 and Bax mRNAs were not affected by 3-min ischemia (data not shown).
Figure 5 (A–C) Dark-field photomicrographs showing Bcl-xL mRNA expression in the hippocampus of sham-operated and 3-min ischemic gerbils: hippocampus of a sham-operated animal (A); hippocampus 1 (B) and 2 (C) d after ischemia. Note a selective increase (more ...)
In the hippocampal CA1 field of sham-operated gerbils, Bcl-xL protein with a molecular mass of approximately 29 kD was constitutively expressed (Fig. E). Forebrain ischemia of 3-min duration caused an apparent decline in Bcl-xL content in the hippocampal CA1 field of gerbils treated with vehicle 1 d after ischemic insult, despite the transient upregulation of Bcl-xL mRNA expression at the same period (Fig. E). These findings suggest that translation of Bcl-xL mRNA is suppressed in the hippocampal CA1 field. On the other hand, IL-3 infusion prevented the decrease in Bcl-xL protein expression 1 d after ischemia (Fig. E). There was also a slight increase in Bcl-xL protein expression in the IL-3–treated hippocampal CA1 region 2 d after ischemia.
Neurotrophic Effect of IL-3 on Cultured Cortical and Hippocampal Neurons.
The above in vivo studies suggest that IL-3 prevents delayed neuronal death in the hippocampal CA1 field through a receptor-mediated expression of Bcl-xL protein. To ascertain this speculation in culture experiments, we investigated (a) whether IL-3 facilitated the survival of cortical and hippocampal neurons, (b) whether cultured cortical and hippocampal neurons expressed IL-3Rα, and (c) whether IL-3 treatment induced the expression of Bcl-xL mRNA and protein in the cultured neurons. Treatment of cultured cortical or hippocampal neurons with IL-3 for 3 d significantly increased the number of surviving neurons compared with the corresponding control culture without IL-3 treatment. MAP2-positive cortical and hippocampal neurons in the IL-3–treated cultures were more numerous than in cultures without IL-3 treatment (Fig. , A–E). Subsequent immunoblot analysis showed that the MAP2-immunoreactive bands of cultured cortical and hippocampal neurons treated with 0.024–15.0 ng/ml of IL-3 (lanes 2–6 in Fig. , F and G) were more intense than those of the control cultures (lane 1 in Fig. , F and G). Densitometric analysis of the MAP2-immunoreactive bands revealed that IL-3 at concentrations of 0.12–15.0 ng/ml enhanced significantly the survival of cultured neurons in a dose-dependent manner (Fig. H). Immunostaining of cultured cortical and hippocampal neurons with the IL-3Rα antibody showed the presence of IL-3Rα in a large population of the neurons (Fig. , I and J). Pretreatment of the IL-3Rα antibody with the homologous antigen abolished the immunoreactions (Fig. K). In contrast to the result of in vivo experiments showing a ligand-induced decrease in IL-3Rα–positive CA1 neurons 4 d after ischemia, we could not detect downregulation of IL-3Rα expression in cultured neurons in response to IL-3 treatment. This might be caused by a large number of CA3 neurons contained in the neuronal culture, because the CA3 neurons exhibited stable IL-3Rα expression.
Figure 6 (A–D) Photomicrographs of MAP2-positive neurons in cultures: cortical (A) and hippocampal (B) neuron cultures without IL-3; cortical (C) and hippocampal (D) neuron cultures treated with 3.0 ng/ml of IL-3. Note that IL-3 significantly increased (more ...)
To investigate the effect of IL-3 on Bcl-xL expression, we first conducted RT-PCR under quantitative conditions using specific primers that amplify a 189-bp fragment of the rat Bcl-xL mRNA. The PCR product showed the expected size, and its identity was confirmed by direct sequencing. Densitometric analysis showed that neurons cultured in the presence of 1 or 10 ng/ml of IL-3 exhibited Bcl-xL mRNA expression approximately three or six times as much as control cultured neurons without IL-3 treatment, indicating that IL-3 upregulated Bcl-xL mRNA expression in a dose-dependent manner (Fig. L). Moreover, IL-3 at concentrations of 0.6–15.0 ng/ml significantly induced Bcl-xL expression in the cultured neurons (Fig. M).
Neuroprotective Effect of IL-3 on Cultured Neurons Exposed to FeSO4.
We investigated whether or not IL-3 attenuated the damage to cortical and hippocampal neurons by FeSO4. Cortical and hippocampal neurons were cultured for 3 d without IL-3 treatment, then FeSO4 was added to the culture medium. The cortical neurons were no longer visible within 2 h in the culture, and hippocampal neurons within 24 h in the culture. MAP2-immunoreactive neurons exposed to FeSO4 (Fig. , A and B) were less numerous than in cultures without FeSO4 treatment (Fig. , A and B). Pretreatment with IL-3 protected cortical and hippocampal neurons against lethal damage caused by FeSO4; MAP2-positive neurons in the IL-3–treated cultures outnumbered those in cultures without IL-3 pretreatment (Fig. , A–E). However, the protective effect of IL-3 on the oxidative damage to neurons by FeSO4 was not observed when IL-3 and FeSO4 were simultaneously added to the cultured medium (data not shown). This suggests that IL-3 protects cultured neurons through induction of intracellular antioxidant agents, including a Bcl-2 family protein(s), rather than by acting alone as a free radical scavenger. Subsequent immunoblot analysis showed that the MAP2 bands of neurons exposed to FeSO4 without IL-3 pretreatment were very thin (lane 1 in Fig. , F and G). In contrast, intense MAP2 bands were detected in neurons in cultures treated with IL-3 at concentrations of 0.12–3.0 ng/ml before the exposure to FeSO4 (lanes 3–5 in Fig. , F and G). The neuroprotective action of IL-3 at different concentrations was quantitatively evaluated by densitometric analysis of the immunoreactive bands (Fig. H). Pretreatment with IL-3 protected significantly cultured neurons against FeSO4-induced damage in a dose-dependent manner. The most effective concentration was 0.6–3.0 ng/ml for hippocampal and cortical neurons (Fig. H). Thus, these in vitro studies suggest that IL-3 exerts a protective effect on cultured neurons through binding to the cell surface receptor, leading to the induction of Bcl-xL protein, which possibly counteracts the neurotoxicity of free radicals.
Figure 7 (A–D) Photomicrographs of MAP2-positive neurons in cultures exposed to FeSO4: cortical (A) and hippocampal (B) neurons in cultures treated only with FeSO4; cortical (C) and hippocampal (D) neurons in cultures pretreated with 3.0 ng/ml of IL-3 (more ...)