Interactions between CB(1) receptors and TRPV1 channels mediated by 12-HPETE are cytotoxic to mesencephalic dopaminergic neurons.

BACKGROUND AND PURPOSES: We recently proposed the existence of neurotoxic interactions between the cannabinoid type 1 (CB(1)) receptor and transient receptor potential vanilloid 1 (TRPV1) channels in rat mesencephalic cultures. This study seeks evidence for the mediator(s) and mechanisms underlying the neurotoxic interactions between CB(1) receptors and TRPV1 in vitro and in vivo. EXPERIMENTAL APPROACH: The mediator(s) and mechanism(s) for the interactions between CB(1) receptors and TRPV1 were evaluated by cell viability assays, immunocytochemistry, Fura-2 calcium imaging, mitochondrial morphology assay, ELISA and Western blot assay in vitro in neuron-enriched mesencephalic cultures. Injections into the substantia nigra and subsequent cell counts were also used to confirm these interactions in vivo. KEY RESULTS: The neurotoxic interactions were mediated by 12(S)-hydroperoxyeicosatetraenoic acid (12(S)-HPETE), an endogenous TRPV1 agonist. CB(1) receptor agonists (HU210 and WIN55,212-2) increased the level of 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), a downstream metabolite of 12(S)-HPETE, which stimulates TRPV1-mediated death of mesencephalic neurons, both in vitro and in vivo. The neurotoxicity was mediated by increased intracellular Ca(2+) concentration ([Ca(2+)](i)) through TRPV1, consequently leading to mitochondrial damage and was attenuated by baicalein, a 12-lipoxygenase inhibitor. CONCLUSION AND IMPLICATIONS: Activation of CB(1) receptors in rat mesencephalic neurons was associated with biosynthesis of 12(S)-HPETE, which in turn stimulated TRPV1 activity, leading to increased [Ca(2+)](i), mitochondrial damage and neuronal death.

Prolonged in vivo gene expression driven by a tyrosine hydroxylase promoter in a defective herpes simplex virus amplicon vector.

A 9.0-kb fragment of the tyrosine hydroxylase (TH) promoter, previously shown to direct tissue-specific expression in transgenic mice, was fused to an Escherichia coli LacZ reporter gene in a defective herpes simplex virus type-1 (HSV-1) amplicon vector (THlac). The HSV immediate early (IE) 4/5 promoter (HSVlac) was used as a control. LacZ gene expression was visualized by X-Gal histochemical and TH immunocytochemical analysis. Two days and 10 weeks after THlac injection into rat caudate nucleus (CN), X-Gal-stained cells were observed in the substantia nigra (SN) and locus ceruleus (LC) ipsilateral to the injection site. These blue cells were TH-positive neurons as evidenced by double labeling with immunocytochemistry. Moreover, the number of X-Gal+, TH+ (double-positive) neurons in the SN increased at 10 weeks as compared to that seen 2 days after THlac injection. In marked contrast, few double-positive nigral neurons were observed either 2 days or 10 weeks after direct injection of THlac into SN. However, neither nigral nor striatal injection of HSVlac resulted in prolonged gene expression. These results suggest that a neuronal, but not a viral, promoter in an HSV vector can produce cell-type-specific, prolonged, and stable gene expression following retrograde transport. In addition, THlac produced infrequent gene expression in TH-negative cells (CN and dorsal to SN) after THlac injection into CN and SN, respectively. Overall, these results suggest that in some in vivo contexts cell-type-preferred expression can be achieved by a cellular promoter in an amplicon vector. Moreover, they underscore the need for the careful and systematic study of neuronal promoters in HSV vectors.

Melatonin increases striatal dopaminergic function in 6-OHDA-lesioned rats.

The purpose of this study was to assess the in vivo effects of melatonin, as an antioxidant, on striatal dopaminergic function in rats with a unilateral 6-hydroxydopamine (6-OHDA) lesion of the striatum. Compared with sham-operated controls and expressed as a ratio relative to the contralateral side, there was an increase in the lipid peroxidation product malondialdehyde (MDA, 142%) and a significant reduction in tyrosine hydroxylase (TH) enzyme activity (28%) and dopamine (DA, 32%) and its metabolite dihydroxyphenylacetic acid (DOPAC, 50%) 2 weeks after 6-OHDA injection. Melatonin treatment almost completely restored MDA levels to normal, suggesting the in vivo action of melatonin as an antioxidant. In parallel, partial, but statistically significant recovery of striatal dopaminergic function, including TH enzyme activity and DA levels, also occurred following melatonin treatment. Taken together with our previous reports showing behavioral and histochemical effects of melatonin on the nigrostriatal dopaminergic system, the present results strongly support the hypothesis that melatonin, as an antioxidant, may have beneficial effects on therapeutic approaches for the treatment of oxidative stress-induced neurodegenerative disease such as Parkinson's disease (PD).

Melatonin protects 6-OHDA-induced neuronal death of nigrostriatal dopaminergic system.

In vivo neuroprotective effects of melatonin on the nigrostriatal dopaminergic system in rats unilateral 6-hydroxydopamine (6-OHDA) lesions were tested. Two weeks after lesioning the dopamine receptor agonist, apomorphine produced rotational asymmetry. In contrast, melatonin treatment significantly reduced the motor deficit following apomorphine challenge. Analysis by tyrosine hydroxylase (TH) immunocytochemistry revealed the loss of cell bodies in the substantia nigra (SN) and absence of terminals in the dorsolateral striatum ipsilaterally. Melatonin treatment also resulted in the survival of dopaminergic neurons in SN and TH-immuoreactive terminals in the dorsolateral striatum. These behavioral and histochemical results may indicate a neuroprotective action of melatonin and suggest a potential pharmacological role in the treatment of Parkinson's disease.

GT1b ganglioside induces death of dopaminergic neurons in rat mesencephalic cultures.

We examined neurotoxicity of GT1b against dopaminergic neurons in vitro. Cultures of mesencephalic cells deprived of serum underwent the loss of 19% of tyrosine hydroxylase immunopositive (TH-ip) neurons. In cultures deprived of serum, treatment with 10-30 microg/ml GT1b attenuated the number of TH-ip neurons by 26-69%, respectively, compared to non-treated cultures. Intriguingly, cultures deprived of serum were more vulnerable to GT1b-induced neurotoxicity. Application of 60 microg/ml GT1b to cultures grown in serum containing media resulted in the loss of 26% of TH-ip neurons, similar to that (28%) observed in serum-deprived cultures treated with 10 microg/ml GT1b. Moreover, in our cultures, absence of nitric oxide (NO) production after GT1b treatment was obvious. The present results strongly suggest direct neurotoxic actions of GT1b against dopaminergic neurons regardless of NO.

Regulation of c-Fos mRNA and fos protein expression in olfactory bulbs from unilaterally odor-deprived adult mice.

Odorant deprivation, produced by unilateral naris closure, profoundly reduces tyrosine hydroxylase (TH) expression within intrinsic olfactory bulb dopamine neurons. The TH gene contains an AP-1 site, which interacts with the product of the immediate early gene, c-fos. c-Fos exhibits activity dependent regulation in the CNS. The hypothesis that odorant stimulation and deprivation might modify c-fos expression in TH neurons was tested in adult CD-1 mice, subjected to unilateral naris closure. After 2 months, naris closed and control mice were exposed to either clean air for 60 min or clean air for 60 min followed by 30 min of alternating exposure to 10% isoamyl acetate (1 min) and air (4 min). A parallel reduction occurred in TH and fos expression (both c-fos mRNA and fos-like immunoreactivity) in the glomerular layer of the odorant-deprived olfactory bulb. Odor stimulation induced a short-lived increase in c-fos mRNA and fos-like immunoreactivity in olfactory bulbs contralateral to naris closure. The increase in fos expression was region-specific in the glomerular layer but more diffuse in mitral and granule cell layers. In olfactory bulbs ipsilateral to naris closure, odor stimulation also induced c-fos mRNA expression in the mitral and granule cell layers and sparsely within limited periglomerular regions. Odor induced expression in mitral and granule cell layers may represent increased centrifugal activity acting on as yet unknown genes. These results suggest a correlation between c-fos mRNA expression and increased neuronal activity in the olfactory bulb which, in turn, acts to regulate TH expression in periglomerular neurons.

Effect of MK801 on cFos-like protein expression in the medial vestibular nucleus at early stage of vestibular compensation in uvulonodullectomized rats.

The purpose of this study was to evaluate the effect of uvulonodullectomy (UNL) on the expression of cFos-like protein (FLP) in the medial vestibular nucleus (MVe) during vestibular compensation and effect of MK801, an N-methyl-D-aspartate (NMDA) antagonist, on FLP expression in the brain stem nuclei at 6 h after unilateral labyrinthectomy (ULX) with UNL in Sprague-Dawley rats. Immunohistochemical staining was performed to visualize FLP in the brain stem nuclei and FLP-positive cells were counted by image analyzer. Lesion-induced asymmetric expression of FLP in the bilateral MVe was observed and maintained up to for 72 h in the ULX group, and 120 h in the UNL + ULX group. Moreover, spatial pattern of FLP expression in the bilateral MVe exhibited the marked difference between the ULX and UNL + ULX groups. MK801 treatment 6 h after ULX showed significant increase in the number of FLP in contralateral MVe (cMVe) of the ULX group, but decrease in cMVe of the UNL + ULX group. These results suggest that the lesion of vestibulocerebellum delays the temporal recovery of FLP expression in MVe and the vestibulocerebellar NMDA receptors relate to FLP expression in MVe.

Role of vestibulocerebellar N-methyl-D-aspartate receptors for behavioral recovery following unilateral labyrinthectomy in rats.

The purpose of current study was to elucidate whether vestibulocerebellar N-methyl-D-aspartate (NMDA) receptors are implicated in MK801 induced vestibular decompensation. Sprague-Dawley rats were unilaterally labyrinthectomized (ULX) and some of them were uvulonodullectomized before ULX (UNL + ULX). Number of spontaneous nystagmus (SN) and degree of head deviation (HD) were used as a parameter of behavioral recovery. MK801 treatment 6 h after ULX produced significant increases in SN and decreased HD in ULX rats, indicating decompensation. In marked contrast, however, MK801 treatment resulted in a great reduction of SN and HD in UNL + ULX rats; suggesting involvement of vestibulocerebellar NMDA receptors in MK801 induced decompensation during early stage of vestibular compensation.

Melatonin protects nigral dopaminergic neurons from 1-methyl-4-phenylpyridinium (MPP+) neurotoxicity in rats.

In the present study, the in vivo neuroprotective effects of melatonin, as an antioxidant, were assessed in Sprague-Dawley rats with a unilateral lesion of substantia nigra (SN) caused by a stereotaxic injection of neurotoxin, 1-methyl-4-phenylpyridinium (MPP+). When expressed as a percentage ratio of lesioned to intact side, increased lipid peroxidation product (malondialdehyde, MDA, 117% of control) and decreased tyrosine hydroxylase (TH) enzyme activity (60% of control) in SN were observed 4 h after MPP+ lesion. In contrast, however, melatonin treatment prevented MPP+ neurotoxicity by the almost complete recovery of MDA (99% of control) and TH levels (96% of control), indicating the potent antioxidative effects of melatonin. In addition, further reduction of TH enzyme activity (52% of control) was seen 1 week after MPP+ infusion. Continuous (twice a day for 5 days), not acute (4 h) treatment with melatonin produced the partial, but not statistically significant, recovery of TH enzyme activity (71% of control), when sacrificed 1 week after MPP+ lesion. Taken together, the present results support the hypothesis that melatonin may provide the useful therapeutic strategies for the treatment of oxidative stress-induced neurodegenerative disease such as Parkinson's disease (PD).

Interleukin-13/-4-induced oxidative stress contributes to death of hippocampal neurons in aß1-42-treated hippocampus in vivo.

AIMS: The present study examined whether Aß(1-42) can induce endogenous expression of interleukin-13 (IL-13) or (IL-4) within activated microglia in the rat hippocampus in vivo. We further investigated whether these cytokines mediate ROS/RNS generation through activation of NADPH oxidase and/or inducible nitric oxide synthase (iNOS), and thus contribute to the degeneration of hippocampal neurons in vivo. RESULTS: Here, we show that IL-13 and IL-4, endogenously expressed in Aß(1-42)-activated microglia in hippocampus in vivo, contribute to degeneration of hippocampal neurons in vivo. Neutralization of IL-13 and IL-4 protected hippocampal neurons in vivo against neurotoxicity by inhibiting activation of microglial NADPH oxidase and iNOS, resulting in attenuation of ROS generation and oxidative damage of protein, lipid and DNA. INNOVATION: To our knowledge, this is the first study to demonstrate the possible involvement of endogenously expressed IL-13 and/or IL-4 in activated microglia after Aß(1-42) injection in the degeneration of hippocampal neurons in vivo. The current findings suggest that the deleterious effects of microglia-derived endogenous IL-13 and/or IL-4 are involved in oxidative stress-mediated neurodegenerative diseases, such as AD. CONCLUSION: We carefully hypothesize that IL-13 and IL-4, well-known as anti-inflammatory cytokines might serve as neurotoxic mediators by enhancing microglia-derived oxidative stress in Aß(1-42)-treated hippocampus in vivo.

Neuroprotective role of microglia expressing interleukin-4.

Little is known about the underlying mechanisms responsible for the death of activated microglia and the functional consequences of the death of these cells, especially in vivo. We show here that intracortical injection of lipopolysaccharide (LPS) led to upregulation of interleukin-4 (IL-4) immunoreactivity, followed by a substantial loss of microglia 3 days later, as visualized by complement receptor type 3 (OX-42) immunostaining and tomato lectin staining. Cells positive for caspase-3 and terminal deoxynucleotidyl transferase mediated fluorescein-dUTP nick-end labeling (TUNEL) were also localized within LPS-activated microglia. IL-4 immunoreactivity was detected as early as 12 hr post-LPS, disappearing at 72 hr. Surprisingly, IL-4 immunoreactivity was detected exclusively in microglia, but not in astrocytes or neurons. In addition, IL-4-neutralizing antibodies markedly increased the survival of activated microglia at 3 days post-LPS. The expression of inducible nitric oxide synthase (iNOS) and tumor-necrosis factor (TNF)-alpha was sustained in parallel in activated microglia, consequently increasing neuronal cell death. To our knowledge, this study is the first to show the endogenous expression of IL-4 in LPS-activated microglia in vivo. Our findings suggest that IL-4 may regulate brain inflammation by inducing the death of activated microglia in vivo and increasing neuronal survival.

Dopamine differentiation factors produce partial motor recovery in 6-hydroxydopamine lesioned rats.

Two-week infusion of muscle-derived differentiation factor (MDF), or human recombinant acidic fibroblast growth factor (aFGF) and/or its muscle-derived activating substance into the striatum of unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rats caused a significant and long lasting (40 days) reduction (48-100%) in amphetamine-induced rotational asymmetry. In parallel with behavioural recovery, striatal tyrosine hydroxylase (TH) activity, dopamine (DA) and dihydroxy-phenyl-acetic acid (DOPAC) levels recovered in a dose-dependent manner in all treated rats when compared to controls. The greatest increments were observed in rats infused with aFGF and its activator. Increases in biochemical indices were not reflected in trophic changes of the dopamine system; thus, the number of TH-immunoreactive neurones and their striatal innervation were unmodified by treatment with MDF. In contrast with the lesioned brain, infusion of these agents into the intact brain produced no change in nigrostriatal dopamine biochemistry. Our results suggest that dopamine differentiation factors may be important in regulating the production of dopamine in the injured brain and, therefore, may be useful in the treatment of DA imbalances associated with certain neurological disorders such as Parkinson's disease.

Dopamine differentiation factors increase striatal dopaminergic function in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned mice.

We have previously shown that muscle-derived differentiation factors (MDF) and human recombinant acidic fibroblast growth factor (aFGF) have beneficial behavioral and neurochemical effects on the nigrostriatal dopaminergic neurons of 6-hydroxy-dopamine (6-OHDA)-lesioned rats (Jin and Iacovitti: Neurobiol Dis 2:1-12, 1995). In the present study, we determined the effects of similar treatments on mice treated with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Five days after unilateral striatal infusion of MDF or aFGF into MPTP-lesioned mice, striatal tyrosine hydroxylase (TH) activity and dihydroxyphenylacetic acid (DOPAC) levels were bilaterally increased (20-35%) compared to untreated (lesion only) or control (phosphate buffered saline + bovine serum albumin) mice. These increases, however, were not accompanied by change in dopamine (DA) levels, indicating an elevation of DA synthesis (TH/DA) and turnover (DOPAC/DA). The present findings that MDF and aFGF may have neurochemical effects in vivo on the lesioned nigrostriatal dopaminergic system suggest their potential pharmacological role in the treatment of Parkinson's disease.

Ischemic damage in the striatum of adult gerbils: relative sparing of somatostatinergic and cholinergic interneurons contrasts with loss of efferent neurons.

The pattern of ischemia-induced cell death was examined with histochemical methods in the striatum of adult gerbils 4 and 7 days after transient forebrain ischemia. The results showed a massive loss of immunoreactivity to enkephalin and tachykinins, peptides present in striatal efferent neurons. In contrast, neurons expressing acetylcholinesterase activity, or choline acetyltransferase immunoreactivity, as well as neurons immunoreactive for somatostatin, were relatively preserved in areas of severe neuronal loss. The selective vulnerability of subpopulations of striatal neurons to transient ischemia in the adult is similar to that observed in the neonate and after local injections of agonists of N-methyl-D-aspartate receptors, but not of agonists of other glutamate receptor subtypes. It also presents striking similarities to the pattern of neuronal death observed in Huntington's disease. The results further support a role for overstimulation of a subtype of excitatory amino acid receptor in ischemia-induced cell death and show that the selective sparing of subpopulations of striatal interneurons after ischemic injury is not related to immaturity of these neurons but also occurs in the adult.

Unique regulation of immediate early gene and tyrosine hydroxylase expression in the odor-deprived mouse olfactory bulb.

Tyrosine hydroxylase (TH), expressed in a population of periglomerular neurons intrinsic to the olfactory bulb, displays dramatic down-regulation in response to odor deprivation. To begin to elucidate the importance of immediate early genes (IEG) in TH gene regulation, the present study examined expression of IEGs in the olfactory bulb in response to odor deprivation. In addition, the composition of TH AP-1 and CRE binding complexes was investigated in control and odor-deprived mice. Immunocytochemical studies showed that c-Fos, Fos-B, Jun-D, CRE-binding protein (CREB), and phosphorylated CREB (pCREB) are colocalized with TH in the dopaminergic periglomerular neurons. Unilateral naris closure resulted in down-regulation of c-Fos and Fos-B, but not Jun-D, CREB, or pCREB, in the glomerular layer of the ipsilateral olfactory bulb. Gel shift assays demonstrated a significant decrease (32%) in TH AP-1, but not CRE, binding activity in the odor-deprived bulb. Fos-B was found to be the exclusive member of the Fos family present in the TH AP-1 complex. CREB, CRE modulator protein (CREM), Fos-B, and Jun-D, but not c-Fos, all contributed to the CRE DNA-protein complex. These results indicated that Fos-B, acting through both AP-1 and CRE motifs, may be implicated in the regulation of TH expression in the olfactory bulb dopaminergic neurons.

Correlation between structure of Bcl-2 and its inhibitory function of JNK and caspase activity in dopaminergic neuronal apoptosis.

To examine the correlation between the structure of Bcl-2 and its inhibitory function of c-Jun N-terminal kinase (JNK) and caspase activity, we established a dopaminergic neuronal cell line, MN9D overexpressing Bcl-2 (MN9D/Bcl-2) or its structural mutants. The mutants comprised a point mutation in the BH1 (G145A; MN9D/BH1) or BH2 (W188A; MN9D/BH2) domain and a deletion mutation in the C-terminal (MN9D/C22), BH3 (MN9D/BH3), or BH4 (MN9D/BH4) domain. As determined by the TUNEL (terminal deoxynucleotidyltransferase nick end-labeling) and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction assay, apoptotic death of MN9D/Neo cells reached 80-90% within 24 h in response to 1 microM staurosporine. Upon staurosporine treatment, JNK activity increased six- to sevenfold over the basal level within 2-4 h. Treatment of MN9D/Neo with both staurosporine and a caspase inhibitor, Z-VAD, attenuated cell death without suppressing JNK activation. Both staurosporine-induced cell death and JNK activation were attenuated in MN9D/Bcl-2. As determined by cleavage of poly(ADP-ribose) polymerase into 85 kDa, Bcl-2 blocked caspase activity as well. When cells overexpressing one of the Bcl-2 mutants were treated with staurosporine, death was attenuated in MN9D/BH1, MN9D/BH2, and MN9D/C22 but not in MN9D/BH3 and MN9D/BH4. Similarly, both JNK and caspase activation were blocked in MN9D/BH1, MN9D/BH2, and MN9D/C22, whereas they were not suppressed in MN9D/BH3 and MN9D/BH4. Taken together, our data indicate that there exists a close structural and functional correlation of Bcl-2 to JNK and caspase activity in staurosporine-induced dopaminergic neuronal cell death.

MPP(+) downregulates mitochondrially encoded gene transcripts and their activities in dopaminergic neuronal cells: protective role of Bcl-2.

The effects of neurotoxins on levels of mitochondrially encoded gene transcripts in a dopaminergic neuronal cell line, MN9D, were examined following treatment with 200 microM N-methyl-4-phenylpyridinium (MPP(+)) or 6-hydroxydopamine (6-OHDA). As confirmed by a Northern blot analysis, levels of cytochrome c oxidase subunit 3 (COX III) and ATPase subunit 6 (ATPase 6) transcript were decreased in a time-dependent manner following treatment with MPP(+) but not with 6-OHDA. Accordingly, enzymatic activity of cytochrome c oxidase (COX) and the intracellular ATP content were also decreased in MPP(+)-treated cells while these remained unaltered in 6-OHDA-treated cells. In the cell death paradigm induced by MPP(+), overexpression of Bcl-2 in MN9D cells (MN9D/Bcl-2) significantly blocked MPP(+)-induced downregulation of COX III and ATPase 6 transcripts. In MN9D/Bcl-2 cells, MPP(+)-induced downregulation of COX activity and the intracellular level of ATP was also blocked. Treatment with a pan-caspase inhibitor, however, neither prevented MPP(+)-induced downregulation of COX activity nor affected intracellular level of ATP in MN9D cells. Taken together, our present data suggest that Bcl-2 may play a regulatory role in energy metabolism by preventing downregulation of mitochondrially encoded gene(s) at a point distinct from its known anticaspase activity in MPP(+)-induced dopaminergic neuronal death.

Cleavage of Bax is mediated by caspase-dependent or -independent calpain activation in dopaminergic neuronal cells: protective role of Bcl-2.

Two cysteine protease families, caspase and calpain, are known to participate in cell death. We investigated whether a stress-specific protease activation pathway exists, and to what extent Bcl-2 plays a role in preventing drug-induced protease activity and cell death in a dopaminergic neuronal cell line, MN9D. Staurosporine (STS) induced caspase-dependent apoptosis while a dopaminergic neurotoxin, MPP(+) largely induced caspase-independent necrotic cell death as determined by morphological and biochemical criteria including cytochrome c release and fluorogenic caspase cleavage assay. At the late stage of both STS- and MPP(+)-induced cell death, Bax was cleaved into an 18-kDa fragment. This 18-kDa fragment appeared only in the mitochondria-enriched heavy membrane fraction of STS-treated cells, whereas it was detected exclusively in the cytosolic fraction of MPP(+)-treated cells. This proteolytic cleavage of Bax appeared to be mediated by calpain as determined by incubation with [(35)S]methionine-labelled Bax. Thus, cotreatment of cells with calpain inhibitor blocked both MPP(+)- and STS-induced Bax cleavage. Intriguingly, overexpression of baculovirus-derived inhibiting protein of caspase, p35 or cotreatment of cells with caspase inhibitor blocked STS- but not MPP(+)-induced Bax cleavage. This appears to indicate that calpain activation may be either dependent or independent of caspase activation within the same cells. However, cotreatment with calpain inhibitor rescued cells from MPP(+)-induced but not from STS-induced neuronal cell death. In these paradigms of dopaminergic cell death, overexpression of Bcl-2 prevented both STS- and MPP(+)-induced cell death and its associated cleavage of Bax. Thus, our results suggest that Bcl-2 may play a protective role by primarily blocking drug-induced caspase or calpain activity in dopaminergic neuronal cells.