INTRODUCTION
Aging is a spontaneous physiological inevitable process,[1 2 3 4 5 6
In recent years, tissue engineering has promoted the development of regenerative medicine aiming to repair, replace, and enhance tissue and organ functions as well as offering therapeutic solutions for many diseases. Adipose-derived stem cells (ADSCs) are mesenchymal stem cells with self-renewal property and multipotential differentiation. ADSCs have the potential to treat various diseases, such as graft-versus-host disease, autoimmune-induced diseases, multiple sclerosis, and diabetes mellitus. ADSCs have become a more acceptable solution for tissue and organ transplantation in regenerative medicine and clinical studies.[7
There are several different routes for delivery of ADSCs to regenerate diseased or injured tissues. They can be administered either by systemic or direct application by injection into the targeted tissue site. The systemic delivery of ADSCs via intravenous, intraperitoneal, intra-arterial, or intracardiac injection is dependent on the homing of ADSCs to the site of disease or injury. Local administration avoids the need of ADSCs to be homed by chemotactic factors, as the ADSCs are directly applied to the targeted tissue.[8
Recent publications have explored the nasal system as a novel stem cell delivery route to the brain. Mesenchymal stem cells (MSCs) delivered into the nasal cavity have been shown to reverse much of the age-related structural damage of the olfactory bulb of rats.[9 10 11
MATERIALS AND METHODS
Animals
Forty-nine female albino Wistar rats were used in this study. Forty-two of them were aged (2-year-old, 200–250 gm) and seven were adults (6-month-old, 150–180 gm). Rats were maintained in the Medical Research Center at the Faculty of Medicine, Ain Shams University Cairo, Egypt, with free access to water and food. They were housed in plastic cages with mesh wire covers and were kept under proper conditions of light, temperature, and humidity.
All animal procedures were carried out according to the guideline of Animal Care and the Scientific Research Ethical Committee of the Faculty of Medicine, Ain Shams University. Permission of Ethical approval was obtained from the Ethical committee of animal research, Faculty of Medicine, Ain Shams University.
Experimental protocol
Animals were kept for 7 days before the beginning of the experiment for acclimatization. Then, they were divided into three main groups:
Group I (adult control group)
Group I included seven adult rats. They received intranasal phosphate-buffered saline (PBS) in a dose of 120 μl (60 μl/nostril). PBS was administered one time at the beginning of the experiment. To ensure complete sniffing of the PBS, the dose was divided into two consecutive doses, 5 min apart (30 μl/nostril in each time). Rats were sacrificed after 15 days from the beginning of the experiment.
Group II (aged group)
Group II included seven aged rats. Rats received PBS in the same dose and route as in Group I. They were also sacrificed after 15 days from the beginning of the experiment.
Group III (aged adipose-derived stem cells-treated group)
Included 35 aged rats that received intranasal PKH26-labeled ADSCs (purchased from the Medical Biochemistry Department, Faculty of Medicine, Cairo University). Seven rats were then sacrificed at each of the following time-points: After 2 h, 1 day, 3 days, 5 days, and 15 days from the beginning of the experiment.
Characterization of adipose-derived stem cells using flow cytometry[12 , 13
Flow cytometry was done to confirm that the used undifferentiated ADSCs maintained their phenotypic characterization after the third passage of growth in culture. Expression of the cell surface markers CD 44, CD45, and CD73 was quantified. Briefly, trypsin-harvested ADSCs were washed with PBS three times, and aliquots of 105 cells were incubated with phycoerythrin-conjugated monoclonal antibodies directed against CD45 (eBioscience Cat # 12-0459), CD73 (BD Pharmingen Cat # 550257) or FITC conjugated monoclonal antibodies directed against CD44 (BD Cat # 348057). Chemicals were purchased from Sigma-Aldrich (2nd St #3306, St. Louis, MO 63118, United States).
Method of intranasal delivery of adipose-derived stem cells
Rats were maintained in a supine position, nonanesthetized. The nasal cavity of each animal was treated with a total of 1 × 106 [9 , 14 9 , 11
Sample collection and tissue preparation
At the end of each time-point, rats were anesthetized by ether inhalation. The heart, kidney, liver, and spleen were collected. Small parts of all specimens (1 cm3 ) were fixed in 10% neutral-buffered formalin for 5 days, after which they were processed to form paraffin blocks.
Immunofluorescent imaging for tracking of the presence of the intranasally administrated PKH26-labelled stem cells in heart, kidney, liver, and spleen tissues was done in Group III. Ten μm-thick paraffin sections were cut and stored flat at 4°C, protected from light until photographed. Deparaffinized, nonstained sections were examined and photographed using LABOMED fluorescence microscope (LX400, cat no: 9126000; New York, USA) in Global Labs center, Cairo, Egypt. Photographing was done using Alexa Fluor 488 (green filter) at low magnification lens (×10).
Paraffin sections of 5 μm thickness were subjected to immunohistochemical staining for the detection of CD105 (Endoglin)-A cell surface marker of ADSCs-in the examined organs in Group III, using Anti endoglin (CD105) polyclonal antibody (Catalog No. R32935) NSJ bioreagents, (MABT117 Sigma-Aldrich).[15
Finally, serial 5 μm-thick paraffin sections were also stained by H and E to evaluate the structural changes of the examined organs in all groups and in all-time intervals.
Morphometric study and statistical analysis
An image analyzer Leica Q win V.3 program installed on a computer in the Department of Histology and Cell Biology, Faculty of Medicine, Ain Shams University, Cairo, was used for the morphometric study for sections of all groups. The computer was connected to a Leica DM2500 microscope (Wetzlar, Germany). The mean area percentage of CD 105 immunohistochemical reaction was measured from five different slides obtained from each animal. Five haphazardly selected nonoverlapping fields were examined for each slide.
All data of morphometric studies were collected and subjected to statistical analysis. The mean value and the standard deviation (SD) were calculated in different groups using SPSS statistical program version 21 (IBM Inc., Chicago, Illinois, USA). Data were statistically analyzed using one-way analysis of variance with post-hoc test for comparison of means. Values were presented as mean ± SD. The significance of the data was determined by P value (probability of chance) where P < 0.05 was considered statistically significant.
RESULTS
Adipose-derived stem cells characterization
Characterization of the cultured cells after the third passage using flow cytometry for CD44, CD73, and CD45 revealed that most cultured cells were positive for CD44, CD73 (shift to the right) and negative reaction was detected for CD45 (shift to the left) [Figure 1 ].
Figure 1: Flow cytometric histograms of rat adipose-derived stem cells are displayed using antibodies directed against CD44, CD45 and CD73. Most cells are positive for CD44, CD73 and negative for CD45
Immunofluorescent microscopy results
As expected, the examination of unstained sections by immunofluorescent microscope showed negative results in all organs of Groups I and II (adult and aged groups respectively) as they were not treated by intranasal labeled stem cells (Group I not shown).
After 2 h of intranasal administration of PKH2-labeled ADSCs, in Group III, green fluorescence was noticed in the heart, kidney, liver, and spleen. At day 1, the ADSCs were seen in most of the heart myocardium; in the renal medulla only, sparing the cortex; in zone 1 around portal areas of the liver; in all areas of the spleen including the capsule. Maximum focal intense fluorescence appeared at day three in all organs and was especially prominent in kidney and liver sections. A gradual decrease in green fluorescence was noticed at day 5, however, remaining apparently high in the renal cortex and medulla and in the splenic capsule as compared to other organs. On day 15, the absence of fluorescence from all organs was demonstrated, except for the spleen that retained low fluorescent intensity in focal sites[Figure 2 ].
Figure 2: Green fluorescence of PKH-26 labelled stem cells from all organs at different time-intervals. Green fluorescence is seen in all organs after two hours from intranasal administration of stem cells. Maximum fluorescence is seen in all organs at day three and day five which is most prominent in kidney and liver. Gradual decrease of fluorescence is noticed at day five which disappear at day 15 from all organs expect the spleen. In aged group, negative reaction is seen in all organs. Immunofluorescence photomicrographs ×100 scale bar: 200 μm
CD105 immunohistochemical study
Examination of immunohistochemically stained sections of the heart of all groups showed positive cytoplasmic CD105 brownish reaction in the endothelial cells of blood capillaries in the myocardium of the aged group (II) and in all time-points of the ADSCs treated group (III). After 2-h of ADCSs intranasal administration, significantly increased (P < 0.05) positive immunoreaction was detected being also present in few cardiomyocytes in focal parts. A significant gradual increase (P < 0.05) in the immunoreaction was also detected in more cardiomyocytes in day 1 and day 3 compared to each other and to the previous time-points, respectively. By day 5, significantly decreased reaction (P < 0.05) was detected-compared to day 3-as very few cardiomyocytes exhibited the brownish cytoplasmic reaction. The disappearance of the reaction from the cardiomyocytes was seen at day 15, being only present in the capillary endothelium. However, it was significantly decreased (P < 0.05) compared to all previous time-points, and nonsignificantly (P > 0.05) different from the aged non-treated group (II) [Figure 3 and Table 1 and Histogram 1].
Figure 3: Immunohistochemical stain for CD105 of different organs at different time-intervals. Insets: positive reaction inside the cells. ([↑]: positive reaction in endothelium of blood capillaries. [▴]: positive reaction inside cells of cardiac myocytes, renal tubules, hepatocytes and cells of the spleen). Immunoperoxidase ×400, insets ×1000, scale bar: 50μm
Table 1: Mean area percentage of CD105 immunohistochemical reaction in different organs at different time-points
Kidney sections of all groups showed a positive cytoplasmic immune reaction in the endothelium of glomerular capillaries and capillaries of the interstitium of the aged group (II) and in all time-points of Group III. Significantly increased positive immunoreaction (P < 0.05) was detected 2-h after ADSCs intranasal administration in cortical and medullary tubular cells. By day one, the reaction was significantly increased (P < 0.05) as compared to the 2-h time-point and to the aged nontreated group (II), appearing in the parietal layer of Bowman's capsule and in more renal tubular cells. The reaction continued to be significantly increased (P < 0.05) on day 3 to be again significantly higher by day 5 as compared to all previous time-points. The minimal reaction appeared in the renal tubular cells and the parietal layer of Bowman's capsule at day 15, in addition to the capillary endothelium. It was significantly decreased (P < 0.05) as compared to all other preceding time-points, and nonsignificantly different (P > 0.05) when compared to the aged Group II.
Examination of the liver from different groups showed positive immune reaction for CD 105 in the endothelium of hepatic sinusoids in Group II and all time-points of Group III. Two hours after intranasal ADCSs administration, few hepatocytes in zone 1 appeared having positive cytoplasmic brownish immunoreaction. Hence, the overall reaction was significantly higher (P < 0.05) as compared to the aged group (II). After one and 3 days, positive immunoreaction was seen in more hepatocytes in zone 1 and few hepatocytes in zones 2 and 3. This reaction was significantly increased (P < 0.05) as compared to their preceding time-points. By day 5, the reaction again significantly increased (P < 0.05) as compared to Group II and to all preceding time-points of Group III. The disappearance of the brownish reaction from the hepatocytes and its retaining in the endothelium of sinusoids was demonstrated on day 15 after the administration of ADSCs. At this time point, it was significantly lower (P < 0.05) as compared to all time-points of Group III, and nonsignificantly increased (P > 0.05) compared to the aged Group II.
Examination of immunohistochemically stained sections of the spleen showed positive CD105 immunoreaction in endothelium of splenic sinusoids of the red pulp of aged group and all time-points of group III. After 2 h, positive immune reaction was detected in a few cells of the white pulp and was significantly increased (P < 0.05) compared to the aged control group (II). This reaction again significantly increased (P < 0.05) and included some parts of the red pulp as well at 1 day, 3 days and 5 days from the administration of ADSCs as compared to Group II and to all their preceding time-points of Group (III). On day 15, the reaction remained in few sites of the white pulp. CD105 immune reaction was significantly decreased (P < 0.05) as compared to all time points of Group III. However, it was still significantly increased (P < 0.05) compared to the aged Group II.
H and E-stained sections
Heart
Light microscopic examination of the H and E-stained heart sections of the control adult group (I) showed striated cardiomyocytes with acidophilic cytoplasm and central oval vesicular nuclei. They were seen with uniform diameter and running in different directions with blood capillaries in-between. In the aged group (II), dilated congested blood capillaries, and inflammatory cells were frequently seen between cardiomyocytes. In ADSCs treated Group III, less dilated congested blood capillaries were seen in all time-points; however, no significant differences were noticed in the cardiomyocytes' structure at all time-points [Figure 4 ].
Figure 4: Structure of myocardium in different groups. ([↑]: oval vesicular nuclei, [▴] blood capillaries between cardiac myocytes, [curved arrow] Pyknotic nuclei) H and E ×400
Kidney
Examination of the H and E-stained kidney sections of control adult group (I) showed the parenchyma of the cortex formed of renal glomeruli, proximal convoluted tubules (PCT) and distal convoluted tubules (DCT). Renal corpuscles were formed of glomerular tufts of capillaries surrounded by Bowman's capsule. The PCTs were seen having narrow lumina and lined with pyramidal cells having acidophilic cytoplasm and basal rounded vesicular nuclei. The DCTs appeared with wider lumina and were lined with low cubical cells having pale acidophilic cytoplasm and rounded nuclei. In the aged group (II), the obliteration of some glomerular capillaries, together with the accumulation of mesangial cells, were noticed in focal areas. The detachment of some tubular cells was detected in most of the tubules. Some of the cells demonstrated pyknotic, karyolytic, or karyorrehectic nuclei. Congestion of blood capillaries was frequently seen in glomeruli and in renal interstitium. After 2 h of intranasal ADSCs administration in group III, similar findings were still seen. These findings were also observed to a lesser extent after 1 day of ADSCs administration. Gradual improvement of the renal structure was noticed on day 3, and day 5 to be notably comparable to the control adult group after 15 days of ADSCs intranasal administration, except for the presence of some congested glomerular capillaries [Figure 5 ].
Figure 5: Kidney sections of different groups. ([*]: glomerular space, [PCT]: Proximal convoluted tubules, [DCT]: Distal convoluted tubules, [V]: Vacuolated cells, [C]: Cast, [↑]: Mesangial cells, [▴]: Congested blood capillaries, [curved arrow]: pyknotic nuclei) H and E ×400
Liver
On examination of the H and E-stained liver sections of control adult group (I), cords of hepatocytes were seen radiating from the central vein in branching and anastomosing pattern. Hepatocytes were polygonal in shape with acidophilic cytoplasm and central rounded vesicular nuclei. Blood sinusoids were noticed as slit-like spaces between hepatocytes. In the aged group (II), some hepatocytes were seen with pyknotic nuclei, and most of the hepatic sinusoids were seen congested. Mononuclear cellular infiltrations were frequently seen between hepatocytes and in the portal tracts. In the intranasal ADSCs treated group (III), no improvement of liver structure could be noticed neither at 2 h nor 1 day. At day 3, many hepatocytes exhibited cytoplasmic vacuolations. The occasional appearance of hepatocytes with dark pyknotic nuclei and few congested sinusoids were detected. On day 5, the hepatic structure showed more improvement with less cytoplasmic vacuolations and less congested sinusoids. After 15 days, the structure of the liver was comparable to control [Figure 6 ].
Figure 6: Liver sections of different groups. ([↑]: rounded vesicular nuclei, [S]: congested hepatic sinusoids, [*]: inflammatory cells, [▴]: pyknotic nuclei, [Δ] vacuolated hepatocytes) H and E ×400
Spleen
Examination of H and E-stained sections of the spleen of the adult control group (I) showed the parenchyma of the spleen surrounded by a capsule and composed of white pulp and red pulp. The white pulp was formed of lymphatic follicles consisting of lymphocytes aggregations with prominent germinal centers and central arteriole. It was separated from the red pulp by the marginal zone consisting of diffuse lymphatic tissue. The red pulp was seen made of splenic cords and sinusoids and focally distributed hemosiderin yellowish-brown granules. In the aged rat group (II), the white pulp follicles appeared larger, irregular with disturbed marginal zone architecture. Paler and larger germinal centers were seen in most of the white pulp as compared to Group I. Dilated marginal sinuses were also evidently seen in focal areas. The disturbed appearance of the red pulp was also observed with an apparently less cellular population dispersed in some frequently seen fibrotic areas. Acidophilic homogeneous exudate was also seen filling focal parts of the red pulp. Apparent increase in hemosiderin content was also noted. Similar findings were seen in the ADSCs treated Group III at 2 h after intranasal administration. Surprisingly, apparently quick improvement of the splenic structure was noticed on day 1 after treatment. This improvement continued through all the time-points that showed eventually a perfect structurally organized spleen comparable to the adult control group by day15 [Figure 7 ].
Figure 7: Spleen sections in different groups. ([WP]: White pulp, [RP]: Red pulp, [GC]: germinal center, [FA]: follicular artery, [*]: pale less cellular parts of red pulp) H and E (adult, aged I and adipose-derived stem cells subgroups ×100) (aged II ×400)
DISCUSSION
Aging and body organs
Aging is a natural biological process that is accompanied by the gradual deterioration of organ's functions owing to the body's inability to combat oxidative stress or maintain homeostasis.[16 17 6 6 1
Adipose-derived stem cells as potential therapeutic agents
This study investigated the ability of ADSCs to be anti-aging agents for multiple body organs. The ADSCs are now well known for their pluripotency and ability to differentiate into mesenchymal and nonmesenchymal lineages.[18 8 , 19 7 , 8 in vitro with lower senescence ratios. Considering clinical applications, ADSCs were reported to be the most suitable source of stem cells owing to the possibility of intravenous transplantation of autologous ADSCs with no immune rejections, ethical problems or tumorigenesis.[18
The International Society for Cellular Therapy and the International Federation of Adipose Therapeutics and Sciences reported that classical cell surface markers of ADSCs include: CD90, CD73, CD105, and CD44, with the absence of expression of CD45 and CD31.[8 , 20 , 21
Intranasal stem cells can reach distant organs
In the current study, examination of immunofluorescent sections for tracking the intranasal administration of ADSCs showed that they reached distant organs other than brain. After 2 h, immunofluorescence was evident in all organs examined, with gradual increase in intensity till reaching its maximum on day three. This could suggest that the administered ADSCs through the intranasal route were directed toward the examined organs in an attempt to heal their age-related structural changes. It was reported that the high vascularity of the respiratory and olfactory epithelium together with its increased surface area due to numerous microvilli, make this region a target for drug absorption to systemic organs. Absorption into the systemic circulation is dependent on crossing both the nasal epithelium and the endothelial lining of the blood vessel. However, the vascular endothelium, not the nasal epithelium, is considered the real barrier.[22 23 24 8
In the present work, anti-CD105 immunohistochemical staining was used to ensure again that the ADSCs homed to the aged organs as it is a classical cell surface marker of ADSCs.[25 , 26 27 , 28 29 18
Potential anti-aging effect of adipose-derived stem cells
In the current study, heart sections did not show structural improvement after intranasal ADSCs administration when compared to the aged group at any time point. Some authors treated myocardial infarction by intramyocardial injection of ADSCs and bone marrow-derived MSCs (BMSCs). ADSCs did not induce angiogenesis as much as BMSCs.[8
The kidney sections showed improvement of some age-related changes after the intranasal administration of ADSCs. It was reported that intravenous ADSCs treatment attenuates renal interstitial fibrosis possibly through inhibition of epithelial mesenchymal transition and inflammatory response.[30
Similarly, the examination of liver sections showed the beginning of the improvement of age-related changes after 3 days from the intranasal administration of ADSCs. It was reported that in partial hepatectomy, hepatocytes accumulate glycogen and lipid to be able to start repair and regeneration.[31 32
Regarding the spleen, the beginning of the improvement of age-related changes occurred at day 5. It was reported that a single ADSCs infusion in rats was accompanied by a considerable number of ADSCs homing to the spleens. In addition, ADSCs infusion promoted anti-inflammatory cytokine IL-10 expression and inhibited pro-inflammatory cytokines IL-6, IL-1 β, and tumor necrosis factor-α expression in the spleen.[33 8 26
It was reported that ADSCs are clinically effective due to their modulation of the host environment, rather than direct effects by cell replacement or differentiation. ADSCs also have antioxidant effects, inhibiting H2 O2 -mediated apoptosis of cells in vitro . The local paracrine effects of ADSCs also occur through the anti-inflammatory and cytoprotective molecules secreted by the transplanted cells.[8 18
Three main mechanisms of MSCs repairing effect have been elucidated. The first mechanism relates to the homing and differentiation of MSCs at injured sites to help to promote healing and regeneration at those injury sites. The second mechanism relates to the production and secretion of cytokines, i.e., growth factors from MSCs that affect other cells in a paracrine manner. The last mechanism is immune modulation, which refers to the ability of MSCs to modulate the immune system via direct interactions with immune cells or via indirect effects mediated by cytokines produced by MSCs.[26
CONCLUSIONS AND RECOMMENDATIONS
ADSCs effectively reached the liver, kidney, spleen, and heart after intranasal administration. ADSCs ameliorated some age-related changes in these organs. Further, in-depth studies will be needed to establish the possibility of repeated administration and an increasing dose of ADSCs. Further studies are also needed to elucidate the detailed molecular mechanisms underlying the changes caused by ADSCs on the aging process. The mechanisms by which the aged myocardium resisted the structural change by the intranasal ADSCs administration is also yet to be clearly explained. Comparative study between the intranasal route and conventional intravenous delivery of stem cells is also needed.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
Acknowledgment
We would like to thank Professor Dr. Nashwa El-Khazragy and her team in Global labs for their kind help in processing immunofluorescent specimens.
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