Some other stimulants, in appropriate doses, can however be neuroprotective. Selegiline: It has been shown to slow early progression of. We review the mechanisms by which hypothermia confers neuroprotection as .. Therapeutic hypothermia consists of 3 phases: induction, maintenance, and. Neuroprotective and disease-modifying effects of the ketogenic diet . A third variation on the diet, known as the Radcliffe Infirmary diet, represents a.
– Neuroprotection 3
Experimental studies have shown that citicoline may indeed increase the synthesis of phospholipids in the CNS [ 97 ] and indicated a neuromodulator effect and a protective role of this molecule on RGC [ 98 ]. In rodent retinal cultures and animal models, citicoline triggered antiapoptotic effects, increased the retinal level of dopamine one of the most important neurotransmitters involved in retinal and postretinal visual pathways [ 99 ], and prevented the thinning of retinal nerve fiber layer [ ].
However, whether dopamine itself works as a neuroprotectant for RGC is not clear yet, since no direct effects of dopamine on RGC survival have been reported. Citicoline has been shown to protect the retina in vivo against kainate-induced neurotoxicity [ ] and to rescue rat RGC following partial optic nerve crush [ ]. A beneficial effect of citicoline oral supplement has been demonstrated in patients with nonarteritic ischemic optic neuropathy.
At the end of the study, PERG, visual evoked potentials, and visual acuity were improved compared to pretreatment values and to a group of patients with no treatment during the same period [ ]. Other clinical studies reported citicoline neurotrophic effects in POAG management [ — ].
Melatonin is a hormone ubiquitously distributed in living systems, from bacteria to plants and animals. The pineal gland is the main source of melatonin, although other organs and cells such as skin, gastrointestinal tract, platelets, and lymphocytes can also make it [ ]. Melatonin receptors MT1, MT2, and to a lesser extent MT3 are consequently found in many tissues [ ], including the eye, where they are well represented in retinal cells [ ] and the ciliary epithelium [ ].
The lipophilic nature of melatonin allows it to easily cross the hematoencephalic and hematoretinal barriers, thus reaching all tissues and the eye with good efficiency in a short time [ ]. Melatonin can affect tissue metabolism and survival via receptor-independent and receptor-dependent mechanisms.
The main receptor-independent activity is due to its strong antioxidant potential. Melatonin is a potent free radical scavenger and antioxidant, different from the other typical antioxidants. In fact, melatonin and its metabolites are able to neutralize numerous toxic oxygen and nitrogen reactive species ROS and NOS, resp. Therefore, melatonin is a more potent antioxidant than vitamins E and C [ ]. Moreover, the large spectrum antioxidant activity of melatonin is potentiated by its regulatory activity on endogenous antioxidant and prooxidant enzymes, upregulating the former and downregulating the latter [ ].
These activities designate melatonin as a neuroprotective agent in several neurodegenerative diseases, in which oxidative damage to neurons is a major player [ ]. In the eye, melatonin has been shown to protect human retinal pigment epithelial cells against oxidative stress [ ] and to slow down photoreceptor degeneration in a mouse model of retinitis pigmentosa [ ]. Moreover, the suppression of melatonin subtype receptor MT1 has been shown to decrease the viability of photoreceptors and RGCs [ , ].
Glutamate accumulation in extracellular spaces can be potentially neurotoxic to the retina [ ], and the impairment of glutamate transporter expression precedes the depression of glutamine synthase activity during ocular pressure loading [ ]. In the hamster retina, it has been shown that melatonin may increase glutamate uptake and glutamine synthase activity, thus decreasing glutamate neurotoxicity [ ].
Melatonin and its analogs have shown hypotonizing effects in both experimental animal models and glaucomatous patients [ — ]. Significant reductions of retinal melatonin levels were found in the rat model of glaucoma induced by chronic ocular hypertension [ ].
The localization of melatonin receptors in the iris and ciliary processes strongly suggests that they are indeed involved in IOP regulation [ , ], most likely through a mechanism that involves the putative MT3 receptors and a local increase in cAMP [ ], similar to what has been described before for forskolin Figure 3 a.
Correspondingly, preliminary clinical observations indicate a cooperative effect on IOP reduction by melatonin and forskolin Pescosolido, personal communication. Hypoxia has also been involved in the development of glaucoma [ — ]. Melatonin has shown neuroprotective effects against hypoxia-induced retinal ganglion cell death in neonatal rats [ ].
Impairment of ocular blood flow is also a relevant player in the etiopathogenesis and progression of the glaucomatous optic neuropathy [ ]. IOP or blood pressure circadian fluctuations cause an unstable oxygen supply, triggering further damage to RGCs [ ]. Melatonin might contribute to the attenuation of these events, both on the IOP and on the blood flow control sides, since it is known to have vasoactive properties and shown to modulate arterial vasoconstriction [ ].
Recognizing its beneficial antioxidant and ocular hypotensive properties, several melatonin related compounds, such as the synthetic analogs and the specific agonists of melatonin receptors, are under investigation [ ]. Among the melatonin analogs, agomelatine is currently attracting interest for its pharmacological activities [ , , , — ].
Agomelatine is a drug used in the treatment of major depressive disorders. In a recent clinical study, the hypotensive activity of oral agomelatine in eyes of POAG patients was revealed: Agomelatine has also shown neuroprotective effects: In vivo treatment with agomelatine reduces the chronic cerebral hypoperfusion responsible for vascular dementia and limits cholinergic dysfunction, oxidative stress, and tissue damage in mice [ ]. The neuroprotective effects of agomelatine and melatonin against NMDA-receptor-mediated white matter lesions have been shown in a newborn mouse experimental model.
Mice that received intraperitoneal agomelatine or melatonin had significant reductions in size of white matter cysts induced by the glutamatergic analog, when compared with controls [ ]. Ginkgo biloba is a native tree of China with various uses in traditional medicine and also as a source of food [ ]. The leaf extract from Ginkgo biloba GBE is rich in biologically active ingredients mainly flavonoids and terpenoids , which can scavenge free radicals and protect cells from lipid peroxidation [ — ].
More interestingly, the polyphenolic flavonoids that are richly present in GBE can act as antioxidants at the mitochondrial level where other antioxidants cannot work , stabilizing mitochondrial membranes and improving their energetic balance specifically in neuronal cells [ , ].
This is an important contribution for glaucoma treatment, since mitochondrial dysfunction has been strongly implicated in POAG pathogenesis [ ]. In several experimental studies, GBE has been shown to exert antioxidant and neuroprotective properties [ — ]. Elevated levels of nitric oxide contribute significantly to the pathogenesis of ocular diseases [ ].
Nitric oxide reacts with superoxides to form peroxynitrites [ ], which cause nitrosylation of cellular proteins, DNA, and lipids, ultimately leading to RGC death [ ]. It was demonstrated in vitro that GBE can scavenge nitric oxide [ ] and possibly inhibit its production [ ].
The protective activity of GBE on isolated rat retinas was evaluated on rats orally treated versus untreated controls with the extract for 10 days. Upon a challenge of the isolated retinas with an oxidant perfusion, GBE contrasted the decrease of ERG-b wave amplitude due to the oxidative damage [ ].
The unstable oxygen supply to the retina and the optic nerve caused by high IOP, blood pressure fluctuations, or disturbed autoregulation also leads to increased oxidative stress, a main contributor to glaucomatous damage [ ]. Beside its antioxidant properties, GBE also shows hemorheological and vasoactive effects, promoting erythrocytes deformability, decreasing fibrinogen levels, and improving blood viscosity and viscoelasticity [ ], and increases microcirculation by improving the endothelium-dependent vasodilation [ ].
Consistently, clinical observation has shown that GBE was able to significantly increase diastolic and systolic velocity in the ophthalmic artery OA of healthy volunteers [ ]. Another clinical study evaluated the effects of GBE in NTG patients, in which vascular dysregulation appears to play a critical role.
In another controlled clinical study on 52 POAG patients, those treated with GBE showed, after 3 months of treatment, a relevant decrease of endothelin-1 ET1, responsible for peripheral vasoconstriction , resulting in increased flow-dependent vasodilation. This was paralleled by a decrease of malondialdehyde-modified low-density lipoproteins and plasma malondialdehyde levels, indicating the activation of an antioxidant response and the attenuation of oxidative stress [ ].
Apoptotic cell death is a hallmark of POAG damage and has been shown at the level of the trabecular meshwork [ ] and the RGC layer [ ]. GBE also shows antiapoptotic properties. Pheochromocytoma cells PC12 treated with GBE were protected from mitochondrial damage induced by serum deprivation or by staurosporine through mechanisms that result in attenuated release of cytochrome-C and less DNA fragmentation, while DNA microarray assay results indicate that transcription of multiple apoptosis-related genes is either up- or downregulated in cells treated with GBE [ ].
Moreover, GBE effects on mitochondria-dependent caspase pathway in cardiomyocytes exposed for 24 hours to hypoxia and four hours to reoxygenation resulted in inhibition of cytochrome-C release from mitochondria, thus decreasing caspase-3 activity and the resulting apoptotic cell death [ ].
Finally, in an experimental in vivo study, it was demonstrated that GBE inhibited the apoptosis of RGC in guinea pigs after optic nerve transection, thus protecting their morphology and function [ ]. Another even more specific agent targeting mitochondria for neuroprotection is the coenzyme Q10 CoQ10 , which is an essential membrane cofactor, with a strong antioxidant activity, in the mitochondrial respiratory chain [ , ].
It also appears to be able to modulate gene expression with anti-inflammatory effects [ ]. In fact, in neurodegenerative diseases, external oxidative stress induces mitochondrial dysfunction, which in turn leads to the increase of ROS generation, and finally leads to apoptotic cell death of the neuronal cells [ ].
CoQ10 has been shown to inhibit ROS generation, to maintain mitochondrial membrane potential during oxidative stress, and to reduce the amount of mitochondrial ROS generation in neuronal cell cultures [ ]. Furthermore, the inhibition of oxidative stress by CoQ10 increases the mitochondrial mass and improves the bioenergetic function in primary optic nerve head rat astrocyte cultures [ ].
High levels of glutamate have been found in the retina of animal models of glaucoma [ , ]. Accordingly, it has been reported that CoQ10 protects retinal cells in vitro against oxidative stress induced by hydrogen peroxide and protects them in vivo after intravitreal injection of N-methyl-D-aspartate [ ]. In a similar experiment, it was demonstrated that CoQ10 results in RGC protection after artificial elevation of extracellular glutamate [ ].
Glaucoma is widely known to be associated with increased RGC apoptosis [ ]. Caspase-7 plays a critical role in this process [ ] since RGCs of mice knocked out for caspase-7 have been shown to be protected from apoptotic death [ ]. More recent data also suggest an important role for Fas receptors and caspasemediated apoptosis in the pathophysiology of glaucomatous neurodegeneration [ ].
Along this line, the antiapoptotic activity of CoQ10 has been evaluated in a rat model of cultured RGCs exposed to external damage and in a mouse model of kainic acid-induced retinal damage. Patients treated with such association showed PERG improvement with consequent enhancement of the visual cortical responses [ ]. Timolol is a nonselective beta-adrenergic receptor antagonist and is one of the main molecules indicated for glaucoma treatment.
Unfortunately, in some cases, adverse cardiovascular effects can occur, and CoQ10 has been shown to be effective in reducing such systemic side effects induced by timolol [ , ]. Polyphenols are secondary plant metabolites generally synthesized from phenylalanine and used by plants in the defense against ultraviolet radiation or aggression by pathogens [ ]. In the last decades, together with the realization that many pathologies, and aging itself, are caused by an excess of oxidative damage, there has been much attention to the health benefits of plant polyphenols mainly those belonging to the class of flavonoids , due to their strong antioxidant properties [ ].
Catechins are flavanols, a subclass of flavonoids. They are the main components of green tea extract, among which epigallocatechin gallate EGCG, also known as epigallocatechingallate is the most abundant.
Catechins may act as radical scavengers, iron chelators, and modulators of prosurvival genes expression and the PKC signaling pathway [ , ]. Along a similar line, it was shown that oral administration of EGCG protects RGCs from degeneration in a mouse model of chronic glaucoma obtained after microbeads injection in the anterior chamber [ ] and in the optic nerve crush rat model [ ]. Intravitreal injection of oxidants such as sodium nitroprusside which generates NO spontaneously triggers significant photoreceptor apoptosis with the rest of the retina relatively unaffected [ , ].
When EGCG is injected into the rat eye together with sodium nitroprusside, its detrimental influence on retinal photoreceptors was attenuated [ ]. EGCG is nongenotoxic, even when administered to animals at doses that are significantly higher than those intended for humans [ ]. Clinical efficacy of a short-term oral supplementation of EGCG has been studied by PERG analysis addressing the electrical activity of RGC , showing that the treatment might favorably influence the inner retinal function in human eyes of glaucomatous patients with early to moderately advanced damage [ ].
Resveratrol belongs to a class of polyphenolic compounds called stilbenes. Some plants notably red grapes produce resveratrol and other stilbenoids in response to stress, injury, fungal infection, or ultraviolet UV radiation [ ].
Besides its own antioxidant activity, more evident in the test tube than in vivo [ ], resveratrol has been shown to induce several antioxidant enzymes, including superoxide dismutase SOD , thioredoxin, glutathione peroxidase-1, heme oxygenase-1, and catalase [ ]. Accordingly, experimental evidence has confirmed that resveratrol has anti-inflammatory, antioxidant, and antiapoptotic activities and a beneficial effect in preventing or slowing down a wide range of age-related diseases [ — ].
The neuroprotective effects of resveratrol dietary supplement were evaluated on the expression of markers for inflammation, oxidative damage, and cellular senescence in primary trabecular meshwork cells exposed to chronic oxidative stress. In another study, the effects of the neuroprotective agents riluzole and resveratrol each with different neuroprotection mechanisms , when administered alone or in combination, were evaluated on the survival of RGC in a rat model of glaucoma. Results indicated that RGCs were significantly preserved in all treatment groups compared to vehicle-only treated control animals and that as expected the association of the two neuroprotective agents gave better results than each one alone [ ].
In a mouse model of mechanical optic nerve injury, long-term diet supplementation with resveratrol has been shown to delay RGC dendrite remodeling and loss [ ]. In another recent work, the hypotensive effect of topical trans -resveratrol was evaluated in rats with steroid-induced ocular hypertension [ ]. The maximum hypotensive effect was obtained with a 0.
The protection of resveratrol in these experiments was associated with the downregulation of the expression levels of matrix metalloproteinase-9, inducible nitric oxide, and heme oxygenase-1 [ ].
Rutin also known as vitamin P or rutoside is a flavonol glycoside found in many plants and fruits [ ], resulting from the combination of the flavonol quercetin and the disaccharide rutinose. Rutin has been shown to possess multiple pharmacological activities [ ]. Two recent clinical studies have shown that oral administration of a food supplement containing rutin potentiates the hypotonizing effects of pharmacological treatments.
Another clinical study showed that oral treatment with an association of forskolin and rutin can blunt the IOP spikes and avoid the damage that may occur after Nd: YAG laser iridotomy for the prevention of primary closed-angle glaucoma [ ]. Furthermore, the association of forskolin and rutin has been shown to improve the symptoms of discomfort in glaucomatous patients suffering from dry eye induced by the long-term use of eye drops preserved with BAK [ ].
Concerning the neuroprotective activity of rutin, different mechanisms have been suggested using both in vivo and in vitro models of neurodegeneration [ — ]. These include the reduction of possibly toxic nitric oxide levels, the inhibition of apoptotic triggers, and the upregulation of neurotrophic factors. In rat pheochromocytoma cells, rutin modulated several neuroprotective genes, including tyrosine hydroxylase, and was able to suppress caspase-3 activity [ ].
Under hypoxic and glutamate stress conditions, rutin significantly increased the survival rate of neonatal rat RGCs by inhibiting the induction of proapoptotic caspase-3 and calpain [ ]. The neuroprotective effects of rutin on the diabetic rat retina have been shown, likely contributed by rutin decreasing both activity and expression of caspase-3 and increasing the protein expression of the survival factor Bcl-2 [ ]. Optimal levels of neurotrophic factors are necessary for normal neuronal functions such as synaptic activity and neuronal survival [ ].
Following incubation with the primary antibodies, slices were incubated for 1 h at RT with a mixture containing the Cy3-conjugated anti-goat Cy3 IgG 1: Briefly, cerebral cortices were dissected, digested with 0.
For cell viability studies see below we used the following experimental conditions: Mixed cortical cell cultures containing both neurons and astrocytes were prepared from fetal mice at 14—16 days of gestation. Cerebral cortices were dissected and centrifuged at 1, g for 7 min. The pellets were resuspended in 1 ml of PM and subjected to a gentle trituration.
The obtained homogenates were combined with appropriate amount of PM four hemispheres in 10 ml of PM for a final cell density of approximately 2. Only mature cultures 13—14 DIV were used for experiments. NaCl, ; KCl, 5. Cell culture media were collected, and cells were harvested by incubation with 1 ml of 0. Trypsinization was stopped by addition of complete medium and the suspension was centrifuged at 1, rpm for 5 min at RT.
Each pellet was washed with cold phosphate buffered saline. Then, tubes were vortexed thoroughly and centrifuged again as above. For this assay, red fluorescence was measured corresponding to the red color of propidium iodide FL-3 detector , and green fluorescence was measured corresponding to the green color of ANX-V FL-1 detector. Both negative control untreated cells and positive control cells treated with a high dose of H 2 O 2 were included in the analysis.
Western blot analysis from lysates of astrocyte cell cultures or whole brains was performed using the following antibodies: After incubation with primary antibodies, filters were washed and incubated with secondary peroxidase-coupled anti goat 1: Statistical analyses were performed as follows: Dickkopf-3 Dkk3 is protective against ischemic neuronal death.
Low and high magnification images are shown. Arrowheads show Dkk3 expression in astrocytes. Protective activity of Dkk3 in cultured astrocytes exposed to oxidative damage or glucose deprivation. B Same as in A but after exposure to 4 h of glucose deprivation. Dkk3 protein levels in cortical cultures of astrocytes after 4 h of glucose deprivation are shown in C. VEGF protein levels in cortical cultures of astrocytes after 4 h of glucose deprivation are shown in D. Phospho-AKT protein levels in cortical cultures of astrocytes after 4 h of glucose deprivation are shown in E.
Bcl2 protein levels in cortical cultures of astrocytes after 4 h of glucose deprivation are shown in F. Dkk3 is protective against excitotoxic neuronal death in mixed cortical cultures.
Ischemic infarct, detected by histological analysis, involved the mid-lateral portion of the cerebral cortex ipsilateral to MCAO Figures 1A,B. We measured infarct volume at three time points 1, 3 and 7 days following MCAO in which ischemic infarct is fully developed under our experimental conditions Mastroiacovo et al. Expression of Dkk3 was assessed by immunohistochemistry at 6 and 12 h post-ischemia, when ischemic damage is partial and near-to-maximal, respectively Mastroiacovo et al.
An increase in Dkk3 immunoreactivity was detected in the medial and inferior portion of the cerebral cortex surrounding the ischemic focus, a region that likely incorporates the ischemic penumbra, at both time points. Double fluorescent staining for GFAP and Dkk3 at this time point indicated that astrocytes largely contributed to the increased expression of Dkk3 at the borders of the ischemic focus Figure 1D , although other cell types also expressed Dkk3 see arrowheads in Figure 1D.
This evidence raised the possibility that induction of Dkk3 in astrocytes could represent a defensive mechanism against ischemic neuronal death. To explore this mechanism in further detail, we examined the protective activity of Dkk3 in primary cultures of astrocytes and in mixed cortical cultures containing both astrocytes and neurons.
In cultures from wild-type mice, glucose deprivation substantially increased Dkk3 protein levels Figure 2C. We extended the analysis to molecules that lie downstream of VEGF receptor activation and likely contribute to cell protection. VEGF receptor activation is known to stimulate the phosphatidylinositolkinase PI3K pathway, which has a widespread role in cell protection mechanisms.
Interestingly, glucose deprivation in astrocytes lacking Dkk3 caused a substantial reduction in phospho-Akt levels, which was not observed in wild-type astrocytes Figure 2E. Activation of the PI3K pathway induces the expression of the antiapoptotic gene, Bcl-2 Pugazhenthi et al. We found a significant reduction in Bcl-2 protein levels in response to glucose deprivation in astrocytes lacking Dkk3, but not in wild-type astrocytes Figure 2F. Moving from this finding, we wondered whether astrocyte cultures prepared from CD1 mice could be more sensitive to a cytotoxic insult.
Neuronal death was assessed 20 h after the NMDA pulse. These findings suggested that Dkk3 produced from astrocytes acted on neighbor neurons to attenuate excitotoxic death. However, the extent of neuronal death varied in different culture preparations, and the appropriate concentrations of NMDA were established from time to time on the basis of data obtained with concentration-response curves of NMDA in sister cultures performed the day before the experiment.
In another experiment, we could also demonstrate that application of hrVEGF 0. We have shown here that: This suggests that endogenous Dkk3 shapes the responsiveness of CNS cells to environmental insults.
Activation of the Wnt pathway is known to support neuronal survival, and induction of the Wnt inhibitor, Dkk-1, contributes to neuronal death in experimental animal models of cerebrovascular disorders Cappuccio et al. Dkk3 differs from all other Dkk family members because its modulatory action on Wnt signaling is complex and context-dependent. In contrast, Dkk3 positively modulates the canonical Wnt signaling pathway in the heart and kidney Federico et al.
On the basis of these findings, we hypothesized the following scenario that could be targeted by therapeutic intervention: While ischemic infarct was greater in mice lacking Dkk3, we have no evidence so far that endogenous Dkk3 is protective against ischemic neuronal damage by a mechanism mediated by VEGF. Excessive VEGF might have detrimental effects in cerebrovascular disorders and age-related neurodegenerative disorders by disrupting endothelial barriers, thereby inducing oedema and inflammation.
It will be interesting to investigate whether exogenous VEGF rescues the pro-ischemic phenotype of Dkk3 mice. However, this task is difficult to achieve because of the pleiotropic effects of VEGF in CNS cells, which require the analysis of different doses of VEGF delivered in different territories of the ischemic damage in different time windows.
Perhaps strategies aimed at enhancing Dkk3 levels might help to overcome these limitations by optimizing the amount of VEGF that is endogenously produced under pathological conditions. As opposed to other members of the Dkk family see Zorn, , the identity of the receptor s that mediate s the biological action of Dkk3 is unknown.
Which of these or other mechanisms mediates the induction of VEGF by Dkk3 in astrocytes remains to be determined. In conclusion, we offered the first evidence that Dkk3 protects cultured neurons and astrocytes through a mechanism that involves the induction of VEGF.
A limitation of the study is that we specifically focused on the involvement of VEGF in the action of Dkk3 and other potential mechanisms have been neglected. In both in vitro and in vivo models, endogenous Dkk3 behaved as a defensive molecule, raising the possibility that strategies aimed at enhancing Dkk3 production are protective.
The analysis of how neurotransmitter receptors expressed by astrocytes control Dkk3 gene expression might be a starting point in the identification of these strategies. CB performed in vitro experiments of glucose deprivation, histological and immunohistochemical analyses, statistical analyses, designed experiments and wrote the manuscript. FB performed in vitro experiments of oxidative stress in cultured astrocytes and western blot analysis.
FM and GBo performed in vivo experiments using the model of permanent focal ischemia. This work was supported by the Italian Ministry of Health Project code:
Advances in Pharmacological Sciences
Caspase 3 activation is essential for neuroprotection in preconditioning. BethAnn McLaughlin, Karen A. Hartnett, Joseph A. Erhardt, Jeffrey J. Legos, Ray F. Dickkopf-3 (Dkk3) is an atypical member of the Dkk family of Wnt inhibitors, which has been implicated in the pathophysiology of neurodegenerative disorders. be reached within about 4 weeks. 8 Typically the increase in. Neuroprotection for the Warrior: Dietary Supplementation. With Omega-3 Fatty Acids. COL Michael.