what volume of o2 at 684 mmhg and 33 ∘c is required to synthesize 16.5 mol of no?

• Periodical List
• J Neurosci
• v.19(12); 1999 Jun 15
• PMC6782637

J Neurosci. 1999 Jun fifteen; 19(12): 5054–5065.

Interleukin-1β Immunoreactivity and Microglia Are Enhanced in the Rat Hippocampus by Focal Kainate Application: Functional Evidence for Enhancement of Electrographic Seizures

Annamaria Vezzani

¹Laboratory of Experimental Neurology and

Mirko Conti

^aneLaboratory of Experimental Neurology and

Ada De Luigi

²Laboratory of Inflammation and Nervous Organisation Diseases, Department of Neuroscience, Istituto di Ricerche Farmacologiche "Mario Negri," 20157 Milan, Italy

Teresa Ravizza

ⁱLaboratory of Experimental Neurology and

Daniela Moneta

¹Laboratory of Experimental Neurology and

Francesco Marchesi

¹Laboratory of Experimental Neurology and

Maria Grazia De Simoni

^twoLaboratory of Inflammation and Nervous System Diseases, Section of Neuroscience, Istituto di Ricerche Farmacologiche "Mario Negri," 20157 Milan, Italia

Received 1998 Jul 31; Revised 1999 Feb 25; Accepted 1999 Mar 22.

Abstract

Using immunocytochemistry and ELISA, nosotros investigated the production of interleukin (IL)-1β in the rat hippocampus after focal application of kainic acid inducing electroencephalographic (EEG) seizures and CA3 neuronal prison cell loss. Next, we studied whether EEG seizures per se induced IL-1β and microglia changes in the hippocampus using bicuculline as a nonexcitotoxic convulsant agent. Finally, to address the functional role of this cytokine, we measured the effect of human recombinant (hr)IL-1β on seizure activeness as i mark of the response to kainate.

Three and 24 hr after unilateral intrahippocampal application of 0.xix nmol of kainate, IL-1β immunoreactivity was enhanced in glia in the injected and the contralateral hippocampi. At 24 hr, IL-1β concentration increased by xvi-fold (p < 0.01) in the injected hippocampus. Reactive microglia was enhanced with a pattern similar to IL-1β immunoreactivity. Intrahippocampal application of 0.77 nmol of bicuculline methiodide, which induces EEG seizures but not cell loss, enhanced IL-1β immunoreactivity and microglia, although to a less extent and for a shorter fourth dimension compared with kainate. Ane nanogram of (hour)IL-1β intrahippocampally injected 10 min before kainate enhanced by 226% the time spent in seizures (p < 0.01). This effect was blocked past coinjection of 1 μg (60 minutes)IL-1β receptor adversary or 0.ane ng of iii-((+)-ii-carboxypiperazin-4-yl)-propyl-one-phosphonate, selective antagonists of IL-1β and NMDA receptors, respectively.

Thus, convulsant and/or excitotoxic stimuli increment the product of IL-1β in microglia-like cells in the hippocampus. In addition, exogenous application of IL-1β prolongs kainate-induced hippocampal EEG seizures by enhancing glutamatergic neurotransmission.

Keywords: bicuculline, cytokines, EEG, epilepsy, interleukin (IL)-1Ra, inflammation, neurodegeneration

The proinflammatory cytokines constitute a group of polypeptide hormones, which were first identified every bit soluble mediators inside the immune system (Schobitz et al., 1994). Recently, cytokines and their receptors take been located in many other tissues, including the peripheral and central nervous systems (Hopkins and Rothwell, 1995). Receptors for interleukin (IL)-1β have been constitute in rodent brain at peculiarly high density in the hippocampus, where they are presumably located on dendrites of granule cells (Takao et al., 1990; Ban et al., 1991; Nishiyori et al., 1997). Both neurons and glia take been shown to produce IL-1β, thus indicating a local source of synthesis in CNS (Benveniste, 1992; Bartfai and Schultzberg, 1993; Hopkins and Rothwell, 1995).

The presence of cytokines in CNS has raised many questions nearly their function and mechanisms of activeness. Besides their well known central actions including effects on the hypothalamo–pituitary–adrenal axis, fever responses, somnogenic effects, and modification of the peripheral allowed response (Schobitz et al., 1994; Hopkins and Rothwell, 1995), some cytokines take been recently shown to affect many neurotransmitters, including noradrenaline, serotonin, GABA, and acetylcholine (Rothwell and Hopkins, 1995; De Simoni and Imeri, 1998), and the expression of diverse neuropeptides and neurotrophic factors in several brain regions (Scarborough et al., 1989; Spranger et al., 1990; Lapchak et al., 1993). In particular, electrophysiological findings take shown that relatively low concentrations of IL-1β, IL-six, and tumor necrosis factor-α (TNF-α) inhibit long-term potentiation (Katsuki et al., 1990; Bellinger et al., 1993; Cunningham et al., 1996), affect excitatory synaptic transmission (Coogan and O'Connor, 1997; D'Arcangelo et al., 1997; Zeise et al., 1997), and modify ionic conductances, particularly Cl⁻ and Ca^two+ currents (Miller et al., 1991;Plata-Salamàn and ffrench-Mullen, 1992).

The involvement of cytokines in neuronal network excitability was recently suggested by the evidence that convulsant drugs increment mRNA levels of IL-1β, IL-6, and TNF-α every bit well as of blazon 2-IL-1 receptor and IL-i receptor antagonist (Ra) in rat forebrain inside hours of seizure induction (Minami et al., 1990, 1991; Nishiyori et al., 1997;Eriksson et al., 1998). In particular, in situ hybridization analysis of IL-1β and IL-1Ra mRNAs later on systemic injection of kainic acid in rats has shown that these transcripts were significantly induced in microglial cells in the hippocampus as well as in other areas of the limbic system (Yabuuchi et al., 1993; Eriksson et al., 1998). Interestingly, autoradiographic analysis of the bounden of radiolabeled IL-ane to rodent brain has revealed a high density of type I receptors in neurons of the dentate gyrus (Takao et al., 1990; Ban et al., 1991), thus suggesting that IL-1β is synthesized in glia and and then secreted in the extracellular infinite for interacting with its specific receptors. In this regard, a larger release of inflammatory cytokines has been described in hippocampal slices of epileptic rats (de Bock et al., 1996).

Evidence in humans also indicates that IL-1 is produced in college amounts in epilepsy, because IL-1α-immunoreactive microglia is enhanced in surgically resected temporal lobe tissue (Sheng et al., 1994).

Although the synthesis of cytokines appears to be tightly regulated at the transcriptional level (Schobitz et al., 1994), the data available so far in experimental models of seizures just analyzed IL-1 mRNA expression, thus non providing direct prove that this cytokine is indeed produced in college corporeality in brain tissue.

In this study, we investigated (i) whether IL-1β production is enhanced in the hippocampus subsequently focal application of kainate inducing both electroencephalographic (EEG) seizures and neuronal cell loss; (two) whether IL-1β production was enhanced in the hippocampus past seizures per se using bicuculline methiodide every bit a nonexcitotoxic convulsant agent; (3) the cell types involved in this effect compared with the design of activated microglial cells, because they have been described equally a major source of IL-1β in CNS (Giulian et al., 1986); and (4) the outcome of intrahippocampal application of (hour)IL-1β on kainate-induced seizures; (five) finally, nosotros probed the hypothesis that glutamate was involved in the effect of (hr)IL-1β on kainate-induced seizures.

MATERIALS AND METHODS

Experimental animals. Male Sprague Dawley rats (250–280 gm) were purchased from Charles River (Calco, Italy), and they were housed at a constant temperature (23°C) and relative humidity (60%) with free access to food and water and a fixed 12 hr calorie-free/nighttime wheel.

Procedures involving animals and their intendance were conducted in conformity with the institutional guidelines that are in compliance with national (4DL N116, GU, suppl xl, xviii-2-1992) and international laws and policies (Europen Community Quango Directive 86/609, OJ L 358, i, Dec 12, 1987; National Institutes of Health Guide for the Care and Use of Laboratory Animals, U.s.a. National Reasearch Council, 1996).

Placement of cannula and electrodes for EEG recordings. Rats were surgically implanted with cannula and electrodes under stereotaxic guidance as described in detail (Vezzani et al., 1986). Briefly, rats were deeply anesthetized using Equithesin (1% phenobarbital and four% chloral hydrate; 3 ml/kg, i.p). Ii screw electrodes were placed bilaterally over the parietal cortex, along with a ground lead positioned over the nasal sinus. Bipolar nichrome wire insulated electrodes (lx μm) were implanted bilaterally into the dentate gyrus of the dorsal hippocampus (septal pole), and a cannula (22 gauge) was unilaterally positioned on top of the dura and glued to 1 of the depth electrodes for the intrahippocampal infusion of drugs. The coordinates from bregma for implantation of the electrodes were (in mm): anteroposterior −3.5; lateral, 2.4; and 3 below dura with the olfactory organ bar gear up at −2.5 (Paxinos and Watson, 1986). The electrodes were connected to a multipin socket (March Electronics, Bohemia, NY) and, together with the injection cannula, were secured to the skull by acrylic dental cement. The experiments were performed 7 d subsequently surgery when the animals did not show any sign of hurting or discomfort.

EEG recordings and intrahippocampal injection of drugs. The procedures for recording the EEG and intracerebral injection of drugs have been previously described (Vezzani et al., 1986). Briefly, the animals were allowed to acclimatize in a Plexiglas cage (25 × 25 × 60 cm) for a minimum of x min before the recording to enable them to adapt to the new environment. The rats were then continued to the pb socket, and a 15–30 min baseline recording was made to establish an adequate control period. EEG recordings (four-channel EEG polygraph, BP8; Battaglia Rangoni, Bologna, Italy) were made continuously during drug injection and upward to 180 min after drug infusion. All the injections were made to unanesthetized rats using a needle (28 gauge) protruding 3 mm below the cannula.

Assay of the EEG. Seizures were induced in rats past intrahippocampal application of nanomole amounts of kainic acid, a glutamate analog acting on kainate-blazon glutamate receptors (Watkins, 1978), or bicuculline methiodide, a GABA-A receptor antagonist (Curtis et al., 1970), and they were measured past EEG analysis. Kainate-induced seizures accept been previously shown to provide a sensitive measure of the anticonvulsant activity of drugs, and they are reportedly associated with neuronal jail cell loss restricted to the CA3 pyramidal cells in the injected hippocampus (Vezzani et al., 1991; Gariboldi et al., 1998). EEG seizures induced by relatively low doses of bicuculline (<ane nmol) are not associated with nerve cell loss, thus representing a nonlesional model of seizure activity (Turski et al., 1985).

The EEG recording of each rat was analyzed visually to observe any activity unlike from baseline. Seizures were defined by the occurrence of discrete episodes consisting of the simultaneous occurrence of at least two of the following alterations in all 4 leads of recordings: high-frequency and/or multispike complexes and/or high-voltage synchronized spike or wave activity. These episodes were typically observed in the start 120 min later kainate injection (Fig.​ 1 b,c). Synchronous spiking was oft observed when seizures subsided (Fig. ​ i d). Seizure episodes after bicuculline were not discrete but in continuity with synchronous spiking (Fig. ​ 1 f,yard). Epileptic-like activity was restricted to the commencement ninety min after bicuculline injection.

Sections of the EEG tracing from a rat injected unilaterally in the dorsal hippocampus with 0.xix nmol of kainic acid (a–d) or 0.77 nmol of bicuculline methiodide (due east–yard). a, e, Baseline recordings;b, c, typical seizures; d, spiking activeness; f, thou, seizure activeness in continuity with synchronous spiking. RCTX, LCTX, Right and left cortex, respectively; RHP, LHP, correct and left hippocampus, respectively. Calibration, 100 μV. Arrowheads inc and d delimit the elapsing of discrete seizure episodes. Fourth dimension elapsed from the injection of convulsant drugs is indicated.

The quantitative parameters chosen to quantify seizure activity after kainate were the latency to the get-go seizure (onset), the total number of seizures occurring in the 3 hour of recording, and the full fourth dimension spent in seizures, which was determined past calculation together the elapsing of all ictal episodes during the EEG recording menses.

In pharmacological experiments, the EEG tracings from rats receiving the diverse drugs and those receiving kainic acid alone were compared visually. Presently after assistants, kainic acid induced stereotyped behaviors such as sniffing and gnawing. "Wet dog shakes" were often observed soon after kainate and during seizures. These behaviors were not significantly affected by the treatments.

Because ictal events and spiking activity were in continuity in rats treated with bicuculline (Fig. ​ 1 f,thou), we included both epileptic events when reckoning the total fourth dimension in seizures (69.half dozen ± 12 min; due north = vi). The onset time to the first epileptic event afterwards bicuculline was ii.6 ± 0.4 min. Jumping and contralateral circling were induced in rats within the first 10 min after injection. Circling and wet dog shakes were oft observed during EEG epileptic-similar activity.

Schedule of treatment. Kainic acid (0.19 nmol in 0.5 μl) or bicuculline methiodide (0.77 nmol in 0.v μl) (Sigma, St. Louis, MO) were dissolved in PBS (0.i m, pH 7.4) or 12% polyethylene glycol (PEG 300; Bracco, Milan, Italy) in PBS, respectively. These were the lowest doses causing EEG seizures in 100% of the animals.

When assessing the upshot of drugs on kainate-induced seizures, rats used as controls were injected with 0.5 μl of rut-inactivated (hr)IL-1β (one ng; homo recombinant IL-1β kindly provided by Dr. Diana Boraschi, Dompé, L'Aquila, Italy; bioactivity in murine thymocyte stimulation assay, ∼3 × 10⁷ U/mg) 10 min earlier kainic acid [0.04 μg (0.19 nmol) in 0.v μl of PBS]. Seizure activity (see Fig. ​ eight, Table ​ 1) did not significantly differ from that measured in rats injected with 0.5 μl of PBS earlier kainate. This excludes endotoxin contamination or unspecific effects on seizure activeness owing to the injection of a big molecule in the hippocampus. (hr)IL-1β was injected at doses ranging from i pg to one ng in 0.5 μl at the aforementioned site as kainic acid 10 min before the convulsant. (60 minutes)IL-1Ra (1 μg; human being recombinant IL-1Ra kindly provided by Dr. Diana Boraschi; inhibitory action in murine thymocyte proliferation assay, 1.seven × 10^vi U/mg), a naturally occurring adversary of type I and type 2 IL-1β receptors (Eisenberg et al., 1990), was injected alone or together with IL-1β in 0.5 μl x min before kainate. (iii-((+)-two-carboxypiperazin-4-yl)-propyl-1-phosphonate) [(R)-CPP; 0.one ng], a selective competitive antagonist of NMDA receptors (Davies et al., 1986), was injected alone or co-injected with 1 ng (hour)IL-1β in 0.v μl 10 min before kainate. All drugs were injected over 1 min using a Hamilton syringe, and an boosted minute elapsed earlier removal of the needle to avoid backflow of the drug through the cannula.

Bar graph showing the effect of IL-1β and/or IL-1Ra on the fourth dimension spent in EEG seizures in rats treated with kainic acid. Control (n = 21) represents rats receiving 0.5 μl of PBS x min earlier 0.19 nmol in 0.5 μl of kainic acid. (60 minutes)IL-1β (due north = 10) was heat-inactivated by boiling the solution for fifteen min. Drugs were unilaterally injected in the hippocampus in 0.five μl alone (n = seven–11) or in combination (n = 9) ten min before kainic acrid. Seizure activity was recorded in freely moving rats for three hr from drug injection. ^a F _(one,51) = eight.6;p < 0.01 by 2-style ANOVA followed by Tukey'due south test for uncounfounded means; *p < 0.01 versus command; °p < 0.01 versus heat-inactivated IL-1β.

Table 1.

Event of (R)-CPP on the IL-1β-induced potentiation of seizure action caused by 0.nineteen nmol of kainic acrid injected in the rat hippocampus

Drug	Dose (ng)	Onset (min)	No. of seizures	Fourth dimension in seizures (min)
Control		11.3  ± 2.0	15.0  ± 1.0	26.three  ± 1.8
IL-1β	i.0	12.iii  ± 2.9	16.0  ± ane.0	52.3  ± four.9*
(R)-CPP	0.1	8.5  ± 1.9	16.0  ± 2.0	26.8  ± iii.iii
IL-1β + (R)-CPP		8.5  ± 1.ix	16.0  ± 3.0	27.0  ± 5.0**,***

After the experiment, all rats treated with kainate and/or the various drugs were killed by decapitation, and their brains were extracted from the skull, rapidly frozen on dry out ice, and sectioned using a cryostat (xl μm) for visual inspection of the traces of the electrodes and the track of the injection needle. Rats treated with bicuculline were transcardially perfused as described beneath. The rats with the electrodes and/or the injection needle out of the hippocampus were excluded from this study.

Tissue preparation for immunocytochemistry. To appraise the changes in the pattern of IL-1β immunoreactivity and for detecting microglia in the hippocampus, rats were injected with kainate or bicuculline methiodide, and their EEG was recorded for 3 hr equally described to a higher place (n = v–8).

For these experiments, kainate was injected in a group of rats unlike from that used for pharmacological studies. Controls (n = 3 each group) were rats implanted with the electrodes and injected with 0.five μl of vehicle. The rats and their respective controls were killed 3 and 24 hr after injection.

Rats were deeply anesthetized with Equithesin and perfused via the ascending aorta with 250 ml PBS, 0.1 m, pH seven.4, followed past 500 ml of chilled paraformaldehyde (iv%) in PBS as previously described (Schwarzer et al., 1996). Afterward carefully removing the brains from the skull, they were post-fixed in the aforementioned fixative as above for 90 min at iv°C and and then transferred to twenty% sucrose in PBS at four°C for 24 hr for cryoprotection. The brains were then rapidly frozen past immersion in isopentane at −seventy°C for 3 min earlier existence sealed into vials and stored at −70°C until use.

Immunocytochemistry. Serial cryostat sections (40 μm) were cut horizontally from all brains. The outset and second sections of each serial of five were collected for staining of IL-1β, and adjacent sections were used for detection of Griffonia simplicifoliaB4-isolectin (GSA I-B4) staining as a specific marking of microglia (Streit, 1990).

Briefly, gratis-floating sections were rinsed for 5 min in 0.4% Triton X-100 in fifty kg Tris-HCl-buffered saline (TBS) at four°C followed by xv min in xx% methanol and 0.six% H_twoO₂ in Triton X-100-TBS. The slices were then incubated at 4°C for 90 min in 10% fetal calf serum (FCS) diluted in 0.iv% Triton X-100-TBS. The master antisera were diluted in 0.four% Triton 10-100-TBS containing 4% FCS, and slices were incubated at 4°C for 72 60 minutes with the principal polyclonal antibody against rat IL-1β (i:500; from Dr. S. Poole, National Found for Biological Standards and Control, Potters Bar, Hertsfordshire, UK). This antibody recognizes both the pro-IL-1β and the mature proteins as assessed by Western blot (S. Pool, personal communication), and its cantankerous-reactivity and specificity have been previously characterized in detail (Bristow et al., 1991; Garabedian et al., 1995). After three 5 min washes in TBS, immunoreactivity was tested by the avidin–biotin–peroxidase technique (Vectastain ABC kit; Vector Laboratories, Burlingame, CA). The sections were so reacted by incubation with 0.4 mm 3′-3′-diaminobenzidine (DAB; Sigma, Munich, Deutschland) in fifty mm Tris-HCl-buffered saline, pH 7.four, and 0.01% H_twoO₂. After DAB incubation, 3 5 min washes were done with TBS, and and so the slices were mounted onto gelatin-coated slides and stale overnight at room temperature. They were dehydrated and coverslipped the adjacent day.

Control slices were prepared using the chief antisera preadsorbed with (60 minutes)IL-ane (i μone thousand, 24 hr, 4°C) and past incubating the slices without the primary antisera.

For assessing microglia, the method described by Streit (1990) was followed. Briefly the sections were immersed in PBS, pH 7.4, for 10 min. Slices were then incubated overnight at 4°C with GSA I-B4 isolectin coupled to HRP (x μg/ml, Sigma) in PBS containing 0.1% Triton X-100, 0.1 grandk CaCl₂, 0.1 mthousand MgCl₂, and 0.1 mmMnCl_two. Later on the overnight incubation, the slices were washed iii times for 5 min in PBS earlier a reaction with DAB, which allows the lectin binding sites to be visualized.

Sections were taken at comparable anteroposterior and mediolateral levels in controls and epileptic rats. Nissl staining was performed with cresyl violet (Paxinos and Watson, 1986) in representative sections of controls and epileptic rats and in a distinct group of rats (northward = 3–4) killed 1 calendar week after convulsant injection to assess neurodegeneration.

ELISA. To quantify the increment in IL-1β depicted by immunocytochemistry, unlike groups of rats were intrahippocampally injected with 0.19 nmol of kainate (north = 5) or 0.77 nmol of bicuculline methiodide (due north = 8) equally previously described.

Twenty four hours later injection, the rats and their controls (implanted with electrodes but injected with 0.5 μl of vehicle;north = 6–8) were killed past decapitation, and their hippocampi were chop-chop dissected out at 4°C and frozen on dry water ice. Brain tissue was weighted and homogenized in ice-cold PBS (5 gm/ml) using a Potter homogenizer (1000 rpm, 10 strokes). The homogenates were centrifuged for 10 min (5000 rpm, iv°C). Ane hundred microliters of the supernatant were taken in duplicate to measure IL-1β.

IL-1β was measured in the hippocampus past a 2-site ELISA using an antibody selective against rat IL-1β (the same used for immunocytochemistry) every bit previously described (De Luigi et al., 1998). Absorbance was read at 405 nm. The detection limit was 3.9 pg/ml.

Statistical analysis of information. Data are the ways ± SE (n = number of animals). The effects of treatments were analyzed by one-style or two-way ANOVA followed by Tukey'due south examination for unconfounded means or by Pupil'due south t test.

RESULTS

Seizure-enhanced immunoreactivity of IL-1β in the rat hippocampus: comparing with activated microglia

Figures ​ ii and ​ 3 depict the pattern of IL-1β immunoreactivity in diverse areas of the injected dorsal hippocampus 3 60 minutes afterward kainic acrid. IL-1β staining in control sections (from rats receiving 0.five μl of PBS) was diffused and barely detectable in glia-like cells throughout the dentate gyrus (Fig.​ 2 A), CA1 (Fig.​ three A), and CA3 (Fig.​ 3 E) areas. Scattered, faintly stained neurons were observed in control sections of CA3 surface area (Fig. ​ three E) and in the granule cell layer (Fig. ​ four A). Seizures enhanced IL-1β immunoreactivity in all regions. Strongly immunoreactive glia-like cells were observed in the granule cells layer and in the hilus (Fig. ​ two C,E) besides as interposed betwixt pyramidal neurons in CA1 (Fig. ​ 3 C) and CA3 (Fig.​ 3 G) areas.

Photomicrographs showing IL-1β immunoreactivity (A, C, E) and B4-isolectin-positive microglia (B, D, F) in coronal sections of the rat dorsal hippocampus 3 hour after a local injection of PBS saline (A, B) or 0.19 nmol of kainic acrid (C–F). E, F, Higher magnifications of pictures respectively depicted inC and D. IL-1β immunoreactivity was markedly increased in glia-similar cells located in the granule cell layer and in the molecular layer (ml) of the dentate gyrus (arrowheads). These cells have a darkly stained prison cell body and branched processes, and some of them take an ameboid shape resembling microglia phenotype (E). Scattered cells with neuronal appearance were also observed (Eastward, pointer). B4-isolectin-positive microglia was also increased in the same regions (D, F). Note that microglia cells and their processes were interposed betwixt granule cells (gc) and CA3 pyramidal neurons in the hilus (h). Calibration confined: A–D, 500 μm;E, F, 200 μm.

Loftier-magnification photomicrographs showing IL-1β immunoreactivity (A, C, East, G) and B4-isolectin-positive microglia (B, D, F, H) in coronal sections of the CA1 (A–D) and CA3 (Eastward–H) areas of the rat dorsal hippocampus iii hr after a local injection of PBS (A, B, E, F) or 0.19 nmol of kainic acid (C, D, G, H). IL-1β-positive neurons were lightly stained in CA3 pyramidal layer and stratum oriens (so) and lucidum (sl) in command sections (Eastward, arrows). IL-1β immunoreactivity and B4-isolectin positive-microglia (circular-shaped cells as well as cells with ramified processes) were enhanced in pyramidal layer (CA1, CA3), stratum oriens and radiatum (sr) of CA1 and CA3 areas, and stratum lucidum CA3 (C, D, Grand, H, arrowheads).f, Fissura hippocampi. Scale bar, 200 μm.

High-magnification photomicrographs showing IL-1β immunoreactivity (A, C) and B4-isolectin-positive microglia (B, D) in coronal sections of the rat dorsal hippocampus 24 hr afterwards a local injection of PBS (A, B) or 0.19 nmol of kainic acid (C, D). Annotation the lengthened pattern of enhanced IL-1β immunoreactivity in glia-like cells (C) and the widespread staining of B4-isolectin-positive microglia (D). Scattered IL-1β immunoreactive neurons were also observed (A, C, arrows). gc, Granule cells; f, fissura hippocampi;CA1, CA1 pyramidal layer; slm, Stratum lacunosum moleculare. Scale bar, 200 μm.

Activated microglia as divers past an ameboid shape and lectin-positive staining was similarly increased in adjacent sections (Figs.​ 2 D,F, ​ 3 D,H). A few round-shaped, darkly stained cells resembling phagocytic cells were observed in the dentate gyrus and CA3 area (Figs. ​ two F,​ 3 H).

Figure ​ 4 depicts the patterns of IL-1β staining (Fig.​ four A,C) and that of activated microglia (Fig.​ 4 B,D) 24 hour after kainic acid in the injected hippocampus. IL-1β-immunoreactive cells and microglia staining were enhanced in the various hippocampal areas with a more diffuse pattern compared with the clusters of heavily stained IL-1β-positive cells and microglia found 3 hr after seizures.

Similar changes were found in the temporal pole of the injected hippocampus. A small increase in IL-1β and microglia staining restricted to stratum radiatum CA3 was observed in the contralateral hippocampus at both fourth dimension points (results not shown).

Nissl staining of coronal sections from rats killed i calendar week after kainate-induced seizures showed the typical pyramidal cell loss restricted to CA3 area equally previously reported (Vezzani et al., 1991) (Fig. ​ 5).

Nissl staining of coronal sections of the dorsal hippocampus of a representative rat i week subsequently the injection of PBS (A) or 0.19 nmol of kainic acid (B). Note the loss of CA3 neurons (B, arrows) that was restricted to the injected dorsal hippocampus. Scale bar, 200 μm.

Rats were injected intrahippocampally with a previously described nonlesional convulsant dose of bicuculline methiodide (Turski et al., 1985) to investigate whether seizures per se, in the absence of cell injury, induced IL-1β and microglia in the hippocampus. Nissl staining performed in coronal brain sections of rats killed ane week after bicuculline-induced seizures (due north = 4) confirmed the lack of neurodegeneration. Thus, the injected and contralateral hippocampi did non differ from vehicle-injected rats as assessed past light microscopic analysis (results not shown).

Figures ​ vi and​ 7 show the design of IL-1β immunoreactivity and activated microglia in the dentate gyrus (Fig.​ 6 A–D) and hippocampus proper (Fig.​ vii A–H) 3 hr after bicuculline injection. IL-1β immunoreactivity was enhanced in all hippocampal subfields compared with vehicle-injected rats, although to a less extent in CA1 and CA3 areas than in kainate-treated rats. Thus, few strongly immunoreactive glial cells were present in CA1 afterward bicuculline (compare Figs.​ 7 C, ​ 3 C). Darkly stained IL-1β-positive cells were absent in CA3, whereas a diffused pattern of faintly stained cells was observed in that location (compare Figs. ​ 7 G, ​ 3 Grand). The changes detected after bicuculline were more pronounced in the injected hippocampus, although they also occurred in the contralateral site and in the temporal poles as observed after kainate injection (results non shown). Xx 4 hours subsequently bicuculline-induced seizures, IL-1β immunoreactivity was very similar to that in vehicle-injected rats, as confirmed by measuring its tissue concentration by ELISA (come across below).

Photomicrographs showing IL-1β immunoreactivity (A, C) and B4-isolectin-positive microglia (B, D) in coronal sections of the rat dorsal hippocampus 3 hr after a local injection of 12.5% PEG (A, B) or 0.77 nmol of bicuculline methiodide (C, D). IL-1β immunoreactivity was markedly increased in glia-similar cells located in the granule cell layer and in the molecular layer (ml) of the dentate gyrus (arrowheads). These cells have a darkly stained cell body and branched processes. Some of these cells were in shut proximity to granule cells (gc) and interposed between CA3c neurons in the hilus (h;C, arrowheads). B4-isolectin-positive microglia was increased in the aforementioned regions (D). Scale bar, 200 μm.

High-magnification photomicrographs showing IL-1β immunoreactivity (A, C, Eastward, Thou) and B4-isolectin-positive microglia (B, D, F, H) in coronal sections of the CA1 (A–D) and CA3 (E–H) areas of the rat dorsal hippocampus 3 hr after a local injection of 12.5% PEG (A, B, E, F) or 0.77 nmol of bicuculline methiodide in PEG (C, D, G, H). IL-1β immunoreactivity was enhanced in darkly stained cells with ramified processes in stratum radiatum (sr) of CA1 pyramidal layer (C, arrowheads). A diffuse blueprint of lightly stained cells was observed in CA3 (Grand, arrowheads). B4-isolectin-positive microglia was enhanced in stratum radiatum (sr) CA1 (D, arrowheads) and in stratum lucidum (sl) CA3 (H, arrowheads). Microglia processes were interposed between pyramidal cells in CA3.f, Fissura hippocampi; so, stratum oriens. Scale bar, 200 μm.

The pattern of activated microglia after bicuculline was similar to that observed after kainate, although cells were less intensively stained in all hippocampal subfields (Figs. ​ vi B,D7B,D,F,H).

ELISA

Using ELISA, an average 16-fold increase in IL-1β concentration was measured in the injected hippocampus 24 hr after kainic acid compared with PBS-injected rats [pg/mg moisture weight tissue: PBS, 1.55 ± 0.15 (n = vi); kainate, 24.two ± 8.4* (due north = v); *p < 0.01 by Student'due southt test]. In accord with the immunocytochemical evidence, a small increase (30%) was also measured in the hippocampus contralateral to the injected site (2.64 ± 1.0 pg/mg), although this effect was not statistically significant.

Twenty iv hours after bicuculline, no meaning differences were found in the injected [pg/mg wet weight tissue: PEG, one.8 ± 0.5 (n = 8); bicuculline, ii.9 ± 1.2] or contralateral hippocampus (bicuculline, 1.24 ± 0.3 pg/mg).

Effect of IL-1β and/or IL-1Ra on kainic acid-induced EEG seizures

Figure ​ 8 shows the outcome of 0.1 and 1.0 ng of (60 minutes)IL-1β and/or ane.0 μg of IL-1Ra on EEG seizure action induced past 0.19 nmol of kainic acid in rats. The intrahippocampal injection of 0.ane ng of (hour)IL1-β 10 min before kainate was ineffective on seizure parameters, whereas i.0 ng of (hour)IL-1β increased past 2.3-fold on boilerplate the time spent in kainate seizures (p < 0.01, one-manner ANOVA followed by Tukey's exam), and this effect was similar to that observed after 10 ng of (hr)IL-1β (information non shown). Interictal activity consisting of high-frequency spiking synchronized in both hippocampi was observed in the 2nd hr of EEG recording later (60 minutes)IL-1β plus kainic acid. (60 minutes)IL-1β did non significantly modify the fourth dimension to onset of seizures (10.8 ± 1.four min; due north = 21) and the number of seizures (xv.0 ± i.0). Heat-inactivated (hr)IL-1β up to i ng did not modify seizures (n = 10).

To assess whether the effect of IL-1β on seizures was receptor-mediated, we injected the animals with 1 μg of (hr)IL-1Ra (Eisenberg et al., 1990). Information technology has been previously reported that doses of IL-1Ra 10²- to 10³-fold college than those of IL-1β are needed to block the functional effects of this cytokine (Arend et al., 1990). This likely depends on the fact that but a few IL-1 receptors need to be stimulated to trigger a biological response; thus high levels of IL-1Ra are necessary for blocking unoccupied receptors (Rothwell, 1991). (60 minutes)IL-1Ra per se (n = 8) did not affect the baseline EEG blueprint simply when co-injected with 1 ng of (60 minutes)IL-1β blocked the proconvulsant event of this cytokine (northward = eight). Thus, the ii.3-fold increment in the full time in seizures induced by ane.0 ng of (hr)IL-1β was abolished in the presence of the antagonist (F _(1,51) = viii.six; p < 0.01, 2-way ANOVA) (Fig. ​ 6). Heat-inactivated (hr)IL-1Ra did non modify the effect of 1 ng of (60 minutes)IL-1β on kainate seizures (data not shown).

Effect of (R)-CPP on the proconvulsant effect of IL-1β

Table ​ 1 shows the upshot of (R)-CPP on the proconvulsant action of (hr)IL-1β on kainate seizures (north = 7). (R)-CPP at 0.1 ng, a dose previously shown to cake NMDA-induced behavioral seizures in rodents (Davies et al., 1986), co-administered with i ng of IL-1β abolished the twofold increment induced past this cytokine in the full time spent in kainate seizures (n = nine;F _(1,30) = 7.6; p < 0.01 by two-fashion ANOVA) without having an issue per se (n = 8). The onset time to seizures and the total number of seizures measured in rats treated with kainic acid (due north = 26) were not significantly modified by 1 ng of (hr)IL-1β (northward = 8) and/or 0.1 ng of (R)-CPP. Selective blockade of NMDA receptors by (R)-CPP did not affect EEG seizures induced by kainic acid per se, as previously reported (Lason et al., 1988; Clifford et al., 1990).

Discussion

In the present study, we provide direct evidence that focal intrahippocampal application of kainic acid in rats inducing EEG seizures and CA3 neuronal damage is associated with a rapid increase in the levels of IL-1β in the hippocampus presumably in activated microglia cells.

The early consecration of IL-1β induced by kainate injection is in accordance with the rapid upregulation of its mRNA after seizure-producing agents (Minami et al., 1990, 1991; Yabuuchi et al., 1993) and with previous evidence showing that the mature course of IL-1β increases as early on every bit 4 hr after intracerebral injection of NMDA in newborn rats (Hagan et al., 1996).

The finding that IL-1β immunoreactivity is enhanced in glial cells resembling activated microglia agrees with the prove that IL-1β mRNA expression induced by systemic kainic acid is localized in glial cells not expressing glial fibrillary acidic protein, a selective marking of astrocytes (Yabuuchi et al., 1993). In addition, recent studies past Andersson et al. (1991) and Taniwaki et al. (1996) have shown that kainate administration activates microglia in brain structures involved in the propagation pathways of hippocampal seizures and closely associated with seizure-induced neuronal damage.

Using bicuculline as a nonlesional model of seizures, we found that both IL-1β and microglia were enhanced in the hippocampus. These changes were induced to a bottom extent and for a shorter elapsing than after kainate, although the time spent in EEG epileptic activity subsequently bicuculline was longer.

These findings bear witness that direct stimulation of the kainate-type of glutamate receptors is not a prerequisite for increasing IL-1β in glia and that seizure activeness per se is sufficient to trigger this outcome. Thus, nosotros found no histological evidence of neuronal cells loss in the hippocampus afterward the relatively low convulsant dose of bicuculline nosotros have used (besides see Turski et al., 1985).

The contribution of neuronal cell injury in CA3 pyramidal layer to the enhanced cytokine response to kainate cannot be solved in the present study. Notwithstanding, various testify suggests that degenerating neurons correspond a stiff betoken for IL-1β consecration (Rothwell, 1991), and microglial cells are known to be rapidly activated in response to even minor pathological changes in CNS (Kreutzberg, 1996).

The widespread pattern of increased IL-1β immunoreactivity observed after focal injection of kainate may depend on different factors or by their concerted activeness: i.e., it is known that IL-1β induces its own synthesis (Dinarello et al., 1987), and this positive feedback loop may contribute to heighten and extend the IL-1 response; mild inflammatory or edemic responses may occur in distant sites because of their direct connections to the injured hippocampus and may trigger IL-1β product; the propagation of epileptic activity from its site of onset to synaptically connected tissue (i.eastward., the contralateral site and the temporal pole of the hippocampus) may trigger IL-1β production per se, equally suggested by our findings after bicuculline seizures.

The mechanisms past which seizure activity per se may induce IL-1β in the resident glial cells are unknown. Protein extravasation in the brain parenchima caused by blood–brain barrier breakup during seizures (Nitsch et al., 1986) and/or ionic changes induced past seizures in the extracellular environment and in glial cells (Barres, 1991) may prime number glia to synthesize higher amounts of cytokines.

In an attempt to address the functional role of this inflammatory cytokine in seizures, we plant that exogenously applied (hour)IL-1β enhanced EEG seizure activity induced past kainic acrid in a dose-dependent and receptor-mediated style. EEG seizures induced by focal kainate injection were not associated with behavioral convulsions, and the increase in focal EEG seizure duration induced by IL-1β had no bear on on rat behavior.

The activity of IL-1β involves an increment in or facilitation of glutamatergic function through the NMDA receptors, because the enhancing effect on seizures was blocked by a selective NMDA receptor antagonist. Blockade of IL-1β outcome by IL-1Ra or (R)-CPP excludes that this cytokine affects seizures past merely delaying kainic acid elimination from the tissue.

The lack of effect of IL-1β on the onset time to seizures may be related to its disability to interfere with the mechanisms involved in seizure consecration (Westbrook and Lothman, 1983), although it is specifically effective on the events that are crucial for seizure maintenance. In this respect, the evidence that IL-1β increases extracellular glutamate availability (Ye and Sontheimer, 1996;Mascarucci et al., 1998) and interacts with NMDA receptor function (as shown by our pharmacological findings) is consistent with its effect of prolonging the time spent in seizures. Thus, both synaptic and intrinsic conductance backdrop of neurons are known to exist involved insustaining synchronized afterdischarges in the hippocampus (Traub et al., 1993). The specific issue of IL-1β on the elapsing of seizures conforms our previous bear witness showing that the onset, number, and duration of EEG seizures tin can be independently affected by drugs (Vezzani et al., 1986, 1991; Gariboldi et al., 1998).

These results are apparently at variance with the electrophysiological bear witness showing that IL-1β has inhibitory furnishings on long-term potentiation (Katsuki et al., 1990; Bellinger et al., 1993; Cunningham et al., 1996; Coogan and O'Connor, 1997), and it augments the GABA-mediated increase in chloride permeability in cortical synaptoneurosomes (Miller et al., 1991). Nevertheless, in vitroand in vivo evidence suggests that the effects of IL-1β on neurons depend on several factors, including the functional land of the neurons (healthy or injured), the timing of cytokine release, the duration of tissue exposure, and the concentration of the cytokine (Rothwell and Hopkins, 1995). In particular, relatively low amounts of IL-1β (picomolar or depression nanomolar range) inhibit neuronal activeness and support neuronal survival (Rothwell, 1991;Morganti-Kossmann et al., 1992), whereas concentrations of IL-1β similar to those used in the present study or higher accept deleterious effects on neuron viability (Rothwell, 1991; Morganti-Kossmann et al., 1992). Interestingly, the functional effects of IL-1β likewise depend on the brain region examined. Thus, the power of this cytokine to modify excitotoxic brain impairment in rats differs betwixt striatum and cortex (Lawrence et al., 1998), and even relatively low doses of IL-1β (0.five northg) consistently decrease synaptic inhibition past ∼thirty% in CA3 pyramidal cells (Zeise et al., 1997).

With regard to the mechanism involved in the facilitation of glutamatergic neurotransmission that appears to mediate the enhancing effect of IL-1β on kainate-induced EEG seizures, it remains to be established whether this is secondary to an altered pattern of neuronal damage (i.e., kainate seizures may exist prolonged past IL-1β promoting impairment, which results in higher glutamate release). In this respect, exogenously practical IL-1β exacerbates excitotoxic neuronal harm and edema induced by ischemia (Yamasaki et al., 1995; Loddick and Rothwell, 1996) as well as the neurodegeneration induced in the cortex by activation of the NMDA and AMPA subtypes of glutamate receptors (Lawrence et al., 1998).

IL-1β, nevertheless, may increase glutamate neurotransmission too past mechanisms independent of neuronal jail cell injury. Thus, IL-1β markedly attenuates astrocytic glutamate uptake (Ye and Sontheimer, 1996). This issue may heighten the extracellular glutamate concentration and may synergize with the increased glutamate release induced by kainate (Ferkany and Coyle, 1983; Young et al., 1988). IL-1β may too direct heighten NMDA receptor role, because IL-1β receptors are associated with signal transduction pathways (i.e., activation of Ser-Thr poly peptide kinase (PK), PKA, and PKC and generation of nitric oxide), which are known to bear on the response of NMDA receptors to endogenous ligands (Hewett et al., 1994; Hollman and Heinemann, 1994;Schobitz et al., 1994).

Finally, the effect of IL-1 β may involve other cytokines as well. Thus, IL-1 induces the synthesis of IL-6 and TNF-α in astrocytes and microglia (Bartfai and Schultzberg, 1993; Schobitz et al., 1994), and many deportment of IL-1 in CNS are mediated by these cytokines (Rothwell, 1991). IL-6 and TNF-α, in turn, have been reported to affect synaptic transmission (Tancredi et al., 1992; Schobitz et al., 1994; Li et al., 1997), and mice overexpressing IL-6 and TNF in glia develop both seizures and neurodegeneration (Campbell et al., 1993; Akassoglou et al., 1997).

The local administration of 0.1–1 μchiliad (hr)IL-1β in the rat hippocampus has been shown to increase the body temperature subsequently 2–4 hr of continuous infusion. This increment was 2°C on average to a higher place physiological values (Linthorst et al., 1994) and was likely mediated by the hypothalamus, because there are reciprocal connections between the hippocampus and diverse hypothalamic areas (Amaral and Witter, 1995). However, it is unlikely that the increase in body temperature plays a role in the result of IL-1β, considering values as high as 42°C are needed to enhance EEG seizure activity induced by kainic acid in rats (Liu et al., 1993).

IL-1Ra alone did non affect kainic acid-induced seizures after its focal application in the hippocampus. Doses of >1 μg could not be tested in our model because of the limit of solubility of this molecule in the small volume required by intrahippocampal administration. Nosotros accept recent testify, withal, that repeated intraventricular injection of 0.1 μg of IL-1Ra significantly reduced EEG seizures caused by intrahippocampal kainate (our unpublished data). Intraventricular injection of IL-1Ra may block more than effectively the activity of endogenously produced IL-1β because of the widespread pattern of induction of IL-1β non solely restricted to the site of intrahippocampal IL-1Ra injection. These findings support a functional role of IL-1β endogenously produced after kainate.

IL-ane is known to upregulate the expression of neurotrophic factors such every bit nerve growth gene and brain-derived neurotrophic factor (Spranger et al., 1990; Lapchak et al., 1993) and neuropeptides such equally somatostatin (Scarborough et al., 1989) that are significantly involved in seizure modulation (Schwarzer et al., 1996; Scharfman, 1997). These events, however, require a time window non compatible with the early activeness of IL-1β in our models of acute seizures. They may represent long-term furnishings of this cytokine and possibly play a functional part in the chronic changes in neurotransmission induced in brain tissue by an acute epileptic effect (Grand. G. De Simoni, C. Perego, T. Ravizza, D. Moneta, Thousand. Conti, Due south. Garattini, and A. Vezzani, unpublished results).

Thus, convulsant and/or excitotoxic stimuli increase the product of IL-1β in microglia-similar cells in the hippocampus. In addition, our pharmacological findings indicate that IL-1β enhances focal electrographic seizures induced by kainate through an increase in glutamatergic neurotransmission. Increased production of IL-1 has been shown in human temporal lobe epilepsy (Sheng et al., 1994), thus suggesting that this cytokine may play a function in the neuropathology of the epileptic tissue.

Footnotes

This work was supported by Telethon Grant East.573. Rat IL-1β ELISA reagents and rat IL-1β antibiotic were kindly provided by Dr. S. Poole. (hour)IL-1β and (hr)IL-1Ra were kindly provided by Dr. Diana Boraschi. We are grateful to Dr. R. Samanin for kindly revising this manuscript and to Andrea Borroni and Carlo Perego for their contribution to part of this study.

Correspondence should be addressed to Dr. Annamaria Vezzani, Laboratory of Experimental Neurology, Istituto di Ricerche Farmacologiche "Mario Negri," Via Eritrea 62, 20157 Milan, Italy.

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Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6782637/

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