Melatonin enhances L-DOPA therapeutic effects, helps to reduce its dose, and protects dopaminergic neurons in 1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine-induced parkinsonism in mice

L-3,4-dihydroxyphenylalanine (L-DOPA) reduces symptoms of Parkinson’s disease (PD), but suffers from serious side effects on long-term use. Melatonin (10–30 mg/kg, 6 doses at 10 hr intervals) was investigated to potentiate L-DOPA therapeutic effects in 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP)-induced parkinsonism in mice. Striatal tyrosine hydroxylase (TH) immunoreactivity, TH, and phosphorylated ser 40 TH (p-TH) protein levels were assayed on 7th day. Nigral TH-positive neurons stereology was conducted on serial sections 2.8 mm from bregma rostrally to 3.74 mm caudally. MPTP caused 39% and 58% decrease, respectively, in striatal fibers and TH protein levels, but 2.5-fold increase in p-TH levels. About 35% TH neurons were lost between 360 and 600 lm from 940 lm of the entire nigra analyzed, but no neurons were lost between 250 lm rostrally and 220 lm caudally. When L-DOPA in small doses (5–8 mg/kg) failed to affect MPTPinduced akinesia or catalepsy, co-administration of melatonin with L-DOPA attenuated these behaviors. Melatonin administration significantly attenuated MPTP-induced loss in striatal TH fibers (82%), TH (62%) and p-TH protein (100%) levels, and nigral neurons (87–100%). Melatonin failed to attenuate MPTP-induced striatal dopamine depletion. L-DOPA administration (5 mg/kg, once 40 min prior to sacrifice, p.o.) in MPTP- and melatonin-treated mice caused significant increase in striatal dopamine (31%), as compared to L-DOPA and MPTP-treated mice. This was equivalent to 8 mg/kg L-DOPA administration in parkinsonian mouse. Therefore, prolonged, effective use of L-DOPA in PD with lesser side effects could be achieved by treating with 60% lower doses of L-DOPA along with melatonin

Astrocytosis in differentiated cells implanted striatum.

<p>Striata, ipsilateral to the side of rotenone infusion were grafted with vehicle without any cells (ROT; A–D), embryonic stem cells undifferentiated (ES; E–H) or 7 days differentiated cells (7 d; I–L), were stained for the astrocyte marker glial fibrillary acidic protein (GFAP) immunoreactivity. Increased immunostaining for GFAP is seen only in the ES and 7 d cells grafted striata. A,E and I are low magnification images, whereas D,H,L and C,G,K are magnified images from the ‘boxes’ each from the previous figures, as marked in the same lane. A,E,I are 5× magnification, whereas scale bars in B,F, and J are 200 µm; in C,G, and K are 50 µm, and D,H, and L are 10 µm.</p

mRNA expression of certain glial markers in the striatum.

<p>(A) Gels showing amplified cDNA bands for different primers. The cDNA was prepared from the mRNA isolated from the control and treated sides of the striatum of the embryonic stem cells, undifferentiated (ES) and 7 days differentiated (7 d) cell transplanted groups. Ratios of the band intensities for GFAP (B), GDNF (C), IBA-1 (D), CD11b-A (E) and CD11b-B (F) as normalised with hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and fold change of band intensities of the treated side compared to the control side was calculated for each animal. Results are presented as Mean ± SEM. *<i>p</i>≤0.05 vs control side of the same group, ^#<i>p</i>≤0.05 vs treated side of the ES transplanted group, n = 3.</p

Co-localization of GFAP and murine specific thymocyte antigen 1.

<p>Striatal sections of 20 µm thickness of embryonic stem cells, undifferentiated (ES; A–H) and 7 days differentiated cells (7 d; I–P) transplanted group were co-stained for glial fibrillary acidic protein (GFAP; B,F,J,N) and murine specific thymocyte antigen 1 (THY1; C,G,K,O) to examine the origin of these glial cells. Figures A–D and I–L are images from locations within the graft, whereas E–H and M–P are locations along the margin of the graft, showing the host tissue and graft. The dotted lines demarcate the margin. GFAP staining is seen outside the graft margin, whereas THY1 positive cells are seen only inside the graft. Scale bar 50 µm.</p

Engraftment of Mouse Embryonic Stem Cells Differentiated by Default Leads to Neuroprotection, Behaviour Revival and Astrogliosis in Parkinsonian Rats

<div><p>We report here protection against rotenone-induced behavioural dysfunction, striatal dopamine depletion and nigral neuronal loss, following intra-striatal transplantation of neurons differentiated from murine embryonic stem cells (mES). mES maintained in serum free medium exhibited increase in neuronal, and decrease in stem cell markers by 7th and 10th days as revealed by RT-PCR and immunoblot analyses. Tyrosine hydroxylase, NURR1, PITX3, LMX1b and c-RET mRNA showed a significant higher expression in differentiated cells than in mES. Dopamine level was increased by 3-fold on 10th day as compared to 7 days differentiated cells. Severity of rotenone-induced striatal dopamine loss was attenuated, and amphetamine-induced unilateral rotations were significantly reduced in animals transplanted with 7 days differentiated cells, but not in animals that received undifferentiated ES transplant. However, the ratio of contralateral to ipsilateral swings in elevated body swing test was significantly reduced in both the transplanted groups, as compared to control. Striatal grafts exhibited the presence of tyrosine hydroxylase positive cells, and the percentage of dopaminergic neurons in the substantia nigra was also found to be higher in the ipsilateral side of 7 days and mES grafted animals. Increased expression of CD11b and IBA-1, suggested a significant contribution of these microglia-derived factors in controlling the limited survival of the grafted cells. Astrocytosis in the grafted striatum, and significant increase in the levels of glial cell line derived neurotrophic factor may have contributed to the recovery observed in the hemiparkinsonian rats following transplantation.</p></div

Expression of NURR1 in 7 and 10 days differentiated cells.

<p>Immunocytochemistry showing the levels of NURR1 proteins as a midbrain dopaminergic neuronal marker in 7days (A–C) and 10 days (D–F) differentiated cells. DAPI stained the nuclei (A,D). Scale bar is 50 µm.</p

Characterization of the grafted cells in the striatum.

<p>Striatal sections of the rotenone-infused control (ROT), embryonic stem cells transplanted animal (ES) and 7 days differentiated cells transplanted animal (7 d) were processed for presence of dopaminergic cells positive for tyrosine hydroxylase (TH) immunoreactivity. Sections were stained with cresyl violet (A, E and I) for localization of neurons (Scale bars for both inset as well as the main frame are 100 µm), and with DAPI for nuclei (B,F,J). Sham transplanted (ROT; B–D); undifferentiated embryonic stem cells transplanted (ES; F–H) and 7 days differentiated cells grafted (7 d; J–L) striatal images are shown. Scale bar is 50 µm for these figures. Scale bar for the magnified images showing TH positive cells, in the right hand corners of the lower panel is 10 µm.</p

Transplantation-induced functional recovery.

<p>Bar graphs in (A) and (B) depict the number of unilateral rotations in hemiparkinsonian rats infused with vehicle only without any cells (ROT), or transplanted with 5×10⁵ embryonic stem cells, undifferentiated (ES) or 7 days differentiated cells (7 d) in the striatum, and treated with amphetamine (Amp) or apomorphine (Apo) respectively on 14th day or 16th days post-transplantation. Amphetamine caused ipsilateral rotations, whereas apomorphine caused contralateral rotations. (C) Preferential bias in body swings, recorded on the 47th day post-lesion or 17th day post transplantation. *<i>p</i>≤0.05 as compared to the pre-transplanted values of rotations, which was similar to vehicle infused group, n = 6–7. (D) Striatal DA levels in the ROT, ES and 7 d groups. *<i>p</i>≤0.05 as compared to the control contralateral side, ^@<i>p</i>≤0.05 as compared to ES transplanted striatum, n = 5–6.</p

Neurons in the nigra of the animals that received unilaterally grafts in the striatum.

<p>Sections passing through the substantia nigra pars compacta were cut and immunostained for tyrosine hydroxylase (arrows; ipsilateral nigra) (A–C) or stained for neurons with cresyl violet (D–F). The sections passing through nigral region of the midbrain of rats that received the vehicle, without cells (A, D), undifferentiated embryonic stem cells (ES; B,E) or 7 days differentiated ES (7 d; C,F). There appeared a significant percentage of improvement in the total number of neurons in the ipsilateral substantia nigra relative to the contralateral side (G), and in TH-positive dopaminergic neurons (H). Results are presented as Mean ± SEM. *<i>p</i>≤0.05, as compared to control or ES transplanted group. Sections from 3 different brain samples were considered for each group.</p

Presence of serotonin and GFAP positive cells in the differentiated cells.

<p>Immunocytochemical localization of serotonin (5-HT; B,C) and glial fibrillary acidic protein (GFAP; E,F) in 7 days differentiated cells showing glial (E) and few serotoninergic neurons (B) in the culture. Scale bar 10 µm.</p