Secretomes of apoptotic mononuclear cells ameliorate neurological damage in rats with focal ischemia

Ethics statement

All animal procedures were approved by the Animal Research Committee of the Medical University of Vienna (Protocol No.: 66.009/127-II/3b/2011) in accordance to the guidelines for the Care and Use of Laboratory Animals by the National Institutes of Health. Efforts were made to minimize suffering.

For the production of human MNC-secretomes, human MNCs were isolated from whole blood of healthy volunteers. This was approved by the ethics committee of the Medical University of Vienna (approval number: EK 2010/034). Participants provided their written informed consent.

Animals

A total of 84 adult male Wistar Rats (Charles River Laboratories, Sulzfeld, Germany) weighing 280–320 g were used. Animals were kept in cages of three to four and accustomed to a 12 hour light-dark cycle for two weeks. Nutrition and tap water were provided ad libitum. Sixteen animals made up the secretome group for which MNCs were extracted out of whole blood and their secretomes produced. Ten animals were randomly selected for the pilot-phase with the intention of establishing protocol and attaining a consistent surgical technique. The remaining 58 animals were used for the study groups and randomly assigned to either the control or the treatment group. Deaths occurred equally in both study groups (total N=21, n=10 in setting 1 [rMNC^{apo sec}] and n=11 in setting 2 [hMNC^{apo sec}], 7 animals were excluded from the study (n=3 in setting 1 and n=4 in setting 2) and euthanized within 6 hours after MCAO due to severe dyspnea and suffering. For statistical analyses, the remaining 30 animals were used (n=16 in setting 1 and n=14 in setting 2) Data were analyzed in a blinded manner. Animals’ tails were marked with colored ink pens before surgery. From that moment on, investigators registered values using only that color code. Surgery was performed by a surgeon unaware if the animals received treatment or placebo. Neuroscore was evaluated by an investigator not involved in the surgical procedure or application of compounds. Statistical analyses were performed by an external statistician.

Production of apoptotic MNC-secretomes from rats (rMNC^{apo sec}) and control medium for experimental setting 1

This section describes the production of apoptotic MNC-secretomes derived from rats (Hoetzenecker et al., 2013). These were used for the treatment group in setting 1 whereas the treatment group in setting 2 received apoptotic MNC-secretomes derived from humans (see below). Syngeneic rat-MNCs were harvested from splenocytes of Wistar rats. For this procedure, animals were anesthetized with an intraperitoneal administration of Ketamine (100 mg/kg) and Xylazine (10 mg/kg). Through a midline incision, spleens were harvested and MNCs were separated by passing spleens through 70 µm and 40 µm cell strainers (BD Biosciences, Vienna, Austria). Red blood cells were lyzed for 90 seconds using a red blood cell lysing buffer (Sigma Aldrich, Vienna, Austria). After washing, MNCs were resuspended in 4 mL CellGro serum-free medium (CellGenix GmbH, Freiburg, Germany) and apoptosis was induced by Caesium-137 irradiation (Department of Transfusion Medicine, Vienna General Hospital) with 45 Gy. Cells were cultivated in CellGro serum-free medium at a concentration of 25×10⁶ cells/mL at 37°C and 5% CO₂ for 18 hours. Cells were removed by centrifugation at 1300 RPM for 9 minutes (Beckman Coulter Allegra^® X-15R, Brea, CA, USA) and cell culture supernatants were then dialyzed against 50 mM ammonium acetate (Sigma Aldrich) using dialysis membranes (cut off: 6–8 kDa; Spectrum laboratories, Breda, The Netherlands) for 24 hours at 4°C on a shaking platter. Subsequently, the dialyzed supernatants were lyophilized over night (Lyophilizator Christ alpha 1–4, Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany). Lyophilization was performed at -20°C and 0.1 mbar pressure. The final product, rMNC^{apo sec}, was stored at -80°C. For the control group, the same cell culture medium that was used for the production of rMNC^{apo sec} was irradiated, cultivated, dialyzed, and lyophilized accordingly. All cell and tissue samples were handled under sterile conditions. Microbial smears on chocolate agars (BD Biosciences, Vienna, Austria), a non-selective media for cultivation of fastidious microorganisms, were performed before lyophilization to rule out contaminations.

Production of apoptotic MNC-secretomes from humans (hMNC^{apo sec}) and control medium according to GMP protocol for experimental setting 2

Pathogen-reduction methods such as photodynamic treatment with methylene blue (MB) plus visible light and gamma radiation have been developed to inactivate viruses and other pathogens in plasma and platelet concentrates. Regulatory authorities require these two pathogen reduction steps for blood derived products such as IVIg, plasma or coagulation factors (Gauvin & Nims, 2010; Lambrecht et al., 1991; Nims et al., 2011; Wallis & Melnick, 1965) to be performed in a good manufacturing practice (GMP) facility.

Human apoptotic MNC-secretomes were prepared as described previously (Lichtenauer et al., 2011). Briefly, venous blood samples (75 mL) were drawn from healthy volunteers (n=15). Blood cells were separated using Ficoll-Paque (GE Healthcare Bio-Sciences AB, Stockholm, Sweden) density gradient centrifugation. MNCs were resuspended with CellGro serum-free medium. After irradiation with 45 Gy as described above, cells were cultivated with CellGro serum-free medium at a concentration of 25×10⁶ cells/mL under sterile conditions for 18 hours. Supernatants were collected by centrifugation and methylene blue (MB) plus light treatment was performed in the Theraflex MB-Plasma system (MacoPharma, Mouvaux, France) using the Theraflex MB-Plasma bag system (REF SDV 0001XQ) and an LED-based illumination device (MacoTronic B2, MacoPharma). Light energy was monitored and reached 120 J/cm². Integrated in the bag system was a pill containing 85 mg of MB, yielding a concentration range of 0.8 to 1.2 mM MB per unit. Removal of MB and photoproducts by Blueflex filtration was done consecutively after treatment. Air was removed from the plasma units before illumination. After lyophilization of this viral inactivated cell culture supernatant as indicated above, the lyophilized powder ran through the second step of pathogen removal, gamma irradiation. Gamma irradiation was performed by Mediscan, which operates a gamma irradiation unit (Gammatron 1500, Mediscan, Seibersdorf, Austria). Gamma rays were generated by radioactive decay of Cobalt 60. For this purpose, apoptotic MNC-secretomes were put in metal sterilization totes that pass on a meandering path through the irradiation vault around the emitting center in five layers. The cobalt unit emits photons that are almost isotropic. Regarding the complex path (280 positions in five layers) the dose is distributed consistently. The dose rate recorded by a Polymethyl methacrylate (PMMA) dosimeter was determined to be 25000 Gy after 23 hours of irradiation. The lyophilized and two-step pathogen-free supernatant of apoptotic MNCs (hMNC^{apo sec}) was stored at -80°C. For the control group, cell culture medium was put through the same steps (cultivation, two step pathogen reduction, irradiation and lyophilization).

Verification of apoptosis in cultured irradiated apoptotic rMNCs using flow cytometry

Syngeneic rMNCs (MNCs derived from rats) were harvested from splenocytes of Wistar rats (n=5) using the same protocol as described earlier. To investigate apoptosis rates during the production of our compounds, washed MNCs that had been cultured for 18 hours were divided into two groups, one to be irradiated in order to induce apoptosis (as described before by using Caesium-137 irradiation with 45 Gy) the other to remain non-irradiated. Except for irradiation, MNCs in both groups were processed equivalently (cultivation, dialysis, lyophilization). Co-staining with Annexin-V/Propidium Iodide (FITC/PI, Becton Dickinson, Franklin Lakes, NJ, USA) was performed following manufacturer’s instructions. The rate of apoptosis was then evaluated on a flow cytometer (FC500, Coulter, CA, USA).

Animal preparation and induction of focal cerebral ischemia

Fifty eight animals were weighed and anesthetized with an intraperitoneal administration of Ketamine (100 mg/kg) and Xylazine (10 mg/kg). This was followed by a subcutaneous injection of Piritramide (15 mg/kg). Animals were then intubated with an 18G intravenous catheter (BD Biosciences) and anesthesia was maintained throughout surgery with 1.5% isoflurane delivered in 1.5 L air and 0.8 L oxygen per minute. Body temperature was regulated using a heating pad (Trixie 76085 Heizmatte, Trixie, Flensburg, Germany). Permanent middle cerebral artery occlusion (MCAO) of the right hemisphere was performed according to the suture model described by Zea Longa et al. using a coated monofilament (Doccol Corporation, CA, USA) (Longa et al., 1989). Briefly, a 3 cm coated monofilament with a thickened tip was inserted into the external carotid artery (ECA) and advanced to the middle cerebral artery (MCA) to induce ischemia in the MCA territory.

Postoperative administration of apoptotic MNC-secretomes

In the first experimental setting, lyophilized rMNC^{apo sec} (produced from 12.5×10⁶ apoptotic rat MNCs) or control medium was resuspended in 0.3 mL saline (Fresenius Kabi, Vienna, Austria) in the laboratory prior to surgery. In order to investigate the potency of apoptotic MNC-secretomes, both the treatment group and the control group (n=29) randomly received 0.3 mL rMNC^{apo sec} or control medium intraperitoneally forty minutes after surgery (Figure 1, blue arrow). In the second setting, lyophilized hMNC^{apo sec} (produced from 12.5×10⁶ apoptotic human MNCs) or lyophilized control medium were each resuspended in the laboratory in 0.3 mL saline prior to surgery. In order to investigate whether a higher dosage and time interval would provide additional benefits, animals from experimental setting 2 (n=29) received two intraperitoneal doses 40 minutes and 24 hours after MCAO induction (Figure 1, red arrows). The rationale for this two-step-approach is given in the discussion. In addition, all animals were given a subcutaneous injection of 3.5 mL/kg saline after surgery and put under a heating lamp until they woke up.

Figure 1. Experimental study setting.

For setting 1, rMNC^{apo sec} (apoptotic MNC-secretomes from rats) were injected 40 minutes after MCAO (blue arrow). In setting 2, hMNC^{apo sec} (apoptotic MNC-secretomes from humans) were administered twice at 40 minutes (0.7 hours) and 24 hours after MCAO (red arrows). In both settings, neurological evaluations were performed at 0 hours (before surgery) as well as 6, 24, and 48 hours after surgery (boxes). Both treatment and control animals were euthanized 48 hours after surgery and brain slices were treated with TTC (2,3,5-triphenyltetrazolium chloride) to stain ischemic areas in the brain.

Measurement of infarct volumes

In both experimental settings, animals were euthanized 48 hours after surgery with an intraperitoneal injection of 600 mg/kg Pentobarbital. Brains were harvested and cut into five 2 mm coronal slices using a brain matrix (Zivic Instruments, Pittsburgh, PA, USA) and razor blades (Zivic Instruments). In order to stain ischemic areas, brain slices were then incubated for 30 minutes at 37°C in a 2% solution of 2,3,5-triphenyltetrazolium chloride (TTC; CarlRoth, Karlsruhe, Germany) (Bederson et al., 1986). Slices were digitalized using a commercially available photo scanner (Epson Perfection V330 Scanner; Figure 2). Lesion volumes were determined by a blinded investigator using ImageJ planimetry software (Version 1.6.0_10; Rasband, W.S., ImageJ, U.S. National Institutes of Health; Bethesda, MD, USA). Lesion volumes were calculated with respect to edema formation using the following formula: 100×(Volume of the contralateral hemisphere-Volume of the ipsilateral hemisphere)/(Volume of the contralateral hemisphere). Ipsi- and contralateral lesion volumes were calculated by multiplication of area with slice thickness summed for all sections (Swanson et al., 1990).

Figure 2. Representative brain slices of rats subjected to MCAO.

Brains were stained with a 2% solution of TTC forty-eight hours after MCAO. Animals received either treatment (in this representative scan: hMNC^{apo sec}) or control medium, in this case, 40 minutes 24 hours after surgery. White areas indicate ischemic tissue while red areas stain for non-ischemic tissue. Animals treated with control medium (left image) had larger ischemic (=white) areas than animals treated with hMNC^{apo sec} (right image).

Neurological evaluation

Neurological examinations were performed by a blinded investigator in both experimental settings using a neurological score before surgery, and 6, 24, and 48 hours after surgery. The test at each time point consists of seven exercises and animals would receive a score ranging from 0 points (no pathological responses) to 7 points (maximum impairment). A successfully completed exercise would add 0 points to the score. Pathological performance in an exercise would add 1 point. The exercises were: left forepaw extension, instability to lateral push from right, tail hanging, walking on ground, whisker movement on the left, hearing, and vision (Nedelmann et al., 2007). E.g. if the rats could not hold against a hand pushing them from the right, they would get 1 point. If they did push against the examiner’s hand, they would get 0 points. Accordingly, if the rats did extend their left forepaw when hauled up by their tail, they would get 0 points. If they could not extend their left forepaw, they would get 1 point. This adds up to a score ranging from 0 points (=no pathological response at all), to 7 points (=highly impaired animal).

Cell culture

Human primary astrocytes, Schwann cells and neurons were obtained from CellSystems (CellSystems Biotechnologie, St. Katharinen, Germany) and cultured in their respective growth medium (CellSystems) at 37°C and 5% CO₂.

Western blot analysis

3×10⁶ astrocytes, Schwann cells and 3×10⁵ neurons were seeded in 6-well plates (Costar, Vienna, Austria) and cultured overnight in their respective growth medium. After removal of the medium, cells were washed twice at room temperature with PBS (Gibco BRL, Gaithersburg, MA, USA) and cultured in their respective basal medium (Astrocyte or Schwann cell growth medium (CellSystems) without growth supplements) for 3 hours. Aliquots of lyophilized human MNC-secretome and control medium were resolved in the different basal media at a 10-fold concentration (lyophilized secretome, derived from 25×10⁶ cells/mL). One tenth of this solution was then directly added to the cell cultures. After 1 hour, the cells were washed at room temperature with PBS and lyzed in 200 µL SDS-PAGE loading buffer (100 mL contain: 1g SDS (Sigma, Vienna, Austria), 3 mg EDTA (Sigma) and 0.75g TRIS (Sigma). pH is adjusted with HCl (Merck, Vienna, Austria) to 6.8 for 10 minutes at room temperature. After sonication (Laborpartner, Vienna, Austria: output = 100%; 20 Cycle for 1 second each) and centrifugation (20000g for 10 minutes) proteins were size-fractionated by SDS-PAGE through an 8 to 18% gradient gel (Amersham Pharmacia Biotech, Uppsala, Sweden) and transferred to nitrocellulose membranes (BioRad, Hercules, CA, USA). Immunodetection was performed with anti-c-Jun (Cell Signaling Technology, Inc. Danvers, MA, USA; 1µg/mL, #9165), anti-phospho-c-Jun (Cell Signaling Technology; 1µg/mL, #9261), anti-CREB (Cell Signaling Technology; 1µg/mL, #9197), anti-phospho-CREB (Cell Signaling Technology; 1µg/mL, #9198), anti-Akt (Cell Signaling Technology; 1µg/mL, #2938), anti-phospho-AKT (Cell Signaling Technology; 1µg/mL, #9271), anti-Erk1/2 (Cell Signaling Technology; 1µg/mL, #4695), anti-phospho-Erk1/2 (Cell Signaling Technology; 1µg/mL, #4376), anti-HSP27 (Cell Signaling Technology; 1µg/mL, #2402), anti-phospho-Hsp27 (Cell Signaling Technology; 1µg/mL, #2404) followed by an HRP-conjugated goat anti-mouse IgG antiserum or a goat anti-rabbit IgG antiserum (GE Healthcare, Freiburg, Germany). Reaction products were detected by chemiluminescence with the ChemiGlow reagent (Biozyme Laboratories Limited, South Wales, U.K.) according to the manufacturer’s instructions.

Neuronal sprouting assay

To investigate neuronal sprouting of human primary neurons, 1×10⁴ cells (CellSystems) were seeded in 24-well plates (Costar) and allowed to adhere for 24 hours. Cells were further cultivated in neuronal medium (see above) without growth factors for five days together with the secretome of hMNC derived from 2.5×10⁶ cells/mL (hMNC^{apo sec}) or control medium. After five days cells were fixed at room temperature in 100% methanol for 10 minutes and stained with methylene blue (Sigma; 0.5% in methanol). Excess methylene blue was washed out with distilled water, and culture wells were evaluated with an inverted microscope (EvosXL, Life Technologies, Carlsbad, CA, USA). Cell cultures were digitalized using an Olympus Digital Camera E-520 (3648×2736 pixels). A blinded observer set a random representative area of the photographed cell culture (as seen in Figure 7b) using Adobe Photoshop Lightroom Software (Version 5.2, 2013; Adobe, San José, CA, USA). (i) on a photograph of cells treated with human apoptotic MNC-secretomes and (ii) on a photograph showing cells treated with control medium. The areas in each photograph measured 1420×2456 pixels. Subsequently, they picked and marked visible distinct, full-length and non-overlapping 30–35 neurites using ImageJ software (Bethesda, MD). Another blinded investigator measured these marked neurons using ImageJ software.

Determination of neurotrophic factors in apoptotic MNC-secretomes and control medium

BDNF, nerve growth factor (NGF) and glial derived neurotrophic factor (GDNF) in rMNC^{apo sec}, hMNC^{apo sec}, and control medium, were measured using commercially available ELISA-kits (Enzyme linked immunosorbent assay; BDNF: catalog# DY248; beta-NGF catalog# DY256; GDNF catalog# DY212; R&D Systems, Minneapolis, MN, USA). All samples were assayed in triplicates. Manufacturer’s instructions were followed and plates were read at 450 nm on a Wallac Multilabel counter 1420 (PerkinElmer, Boston, MA, USA).

Determination of BDNF in rat plasma

To see whether intraperitoneal administration of apoptotic MNC-secretomes and control medium influence BDNF production, six rats were injected intraperitoneally with hMNC^{apo sec} (n=3), or control medium, (n=3). Rats were euthanized with an intraperitoneal injection of 600 mg/kg Pentobarbital 24 hours after injection with hMNC^{apo sec} (secretomes of 12.5×10⁶ cells) or control medium. Blood was retrieved in heparinized tubes, centrifuged, and plasma was stored at -20°C. Rat BDNF levels were measured using a commercially available BDNF-ELISA (catalog# KA0330, Abnova, Taipei, Taiwan) and plates were read at 450 nm on a Wallac Multilabel counter 1420 (PerkinElmer, Boston, MA, USA).

Statistical analyses

To test for differences in lesion volume between control and treatment groups the Mann-Whitney U-test was applied. Differences were also assessed graphically using box-plots. The calculations were performed separately for each experimental setting. Linear mixed models were calculated to explain neuroscores by time point, treatment and interaction effects between time point and treatment. A random intercept term was included for each individual animal to account for the correlation of observations within an individual. The calculations were done using the MIXED procedure in SAS 9.3. The neuroscores were also assessed graphically by plotting a time curve of mean neuroscore values±SD for each group. Neurite lengths in neuronal cultures were compared applying the student’s t-test. A p-value of 0.05 or below was considered significant.

Apoptosis rates in irradiated apoptotic rMNCs

In order to see the extent of apoptosis in rMNCs that had been cultured for 18 hours, we analyzed apoptosis rates in cultured irradiated and cultured non-irradiated rMNCs from 5 donors using flow cytometry. Analysis of cultured irradiated rMNCs revealed apoptosis rates of 85±5% (mean±SD) while 12±5 did not stain for FITC/PI, thus being viable. In the non-irradiated rMNC control group, 44±7 cells were apoptotic with 55±7 still being viable (Figure 3).

Figure 3. Apoptosis rates in irradiated and non-irradiated cultured rMNCs.

Mean percentage values of viable (white) and apoptotic (red) rMNCs are given in this bar graph. The percentage of cells in either state are shown for non-irradiated rMNCs (control) and irradiated rMNCs after 18 hours in cell culture. Irradiated rMNCs correspond to the compound we used throughout the study.

Apoptotic MNC-secretomes reduce the infarction volume in an experimental MCAO model

In a rat model of MCAO we examined the potential of apoptotic MNC-secretomes to reduce ischemic lesion volumes in an allogeneic (experimental setting 1, rMNC^{apo sec}) and a xenogeneic approach (experimental setting 2, hMNC^{apo sec}) (Figure 1). The results of the allogeneic setting displayed significantly lower lesion volumes in the treatment group compared to the control group as shown by TTC-staining (Figure 4). Treatment with rMNC^{apo sec} led to a mean decrease of 36% in total infarct volume. Hemispheric lesion volumes (mean±SD) in the control group were 59%±8% ranging from 50% to 73% (Mann Whitney U-test; * p=0.0006; Figure 4a). The treatment group had a mean hemispheric lesion volume of 38%±11% ranging from 24% to 51%. In the xenogeneic setting, we injected hMNC^{apo sec} 40 minutes and 24 hours after MCAO. The reduction of the infarction volume in the xenogeneic setting was statistically significant and comparable to that observed in the allogeneic setting (Mann Whitney U-test; * p=0.0041; Figure 4b). The mean decrease in total infarct volume was 37%. Hemispheric lesion volumes (mean±SD) in the control group were 52%±8% ranging from 42% to 67%. The treatment group had a mean hemispheric lesion volume of 33%±11% ranging from 21% to 48%.

Figure 4. Infarct volumes in control animals and animals treated with apoptotic MNC-secretomes.

The percentage of hemispheric lesion volumes (%HLV) are represented as box and whiskers plots, wherein the boxes indicate the 1^st and the 2^nd quartile and the whiskers the minimum and maximum within 1.5 times the interquartile range from the box. Figure 4a shows lesion volumes as the extend of ischemia in setting 1, where apoptotic MNC-secretomes derived from rats were administered 40 minutes after MCAO compared to controls. Figure 4b corresponds to setting 2, where apoptotic MNC-secretomes derived from humans were administered 40 minutes and 24 hours after MCAO. In both settings, MNC^{apo sec} (red boxes) caused a significant decrease in infarct volumes (* p=0.0006 for setting 1, Figure 4a, and * p=0.0041 for setting 2, Figure 4b) compared to the control group (white boxes) that received only cell culture medium.

Apoptotic MNC-secretomes improve neurological outcome in an experimental MCAO model

In order to discover the effects of apoptotic MNC-secretomes on the neurological outcome, we performed a neurological exam on each animal at 4 specific time points. The first score was measured prior to surgery (baseline; 0 hours) and was 0 points for all animals. This was followed by 3 postoperative measurements at 6, 24 and 48 hours. All animals expressed neurological stroke-symptoms immediately after anesthesia wore off. The fixed effect coefficient estimates of the mixed models, their standard errors and p-values are shown in Table 1a and Table 1b. The factor Treatment was coded in a way that Treatment=0 corresponds to the control group and Treatment=1 corresponds to the treatment group. The time point 6 h is the reference group for the factor Time.

The results from both settings are similar. At time point 6 h there is no significant mean difference in the neuroscores between control and treatment, which is suggested by the coefficient for Treatment. The coefficients for Time 24 h and Time 48 h are not significantly different from zero. We can therefore not conclude that the mean neuroscore in the control group changes with time. The interaction terms for Treatment and Time, however, are significant. This indicates a significant decrease in the mean neuroscore over time in the treatment group. These results correspond well to the graphical depictions of the time curves (Figures 5a and 5b).

Figure 5. Neurological outcome score in control animals and animals treated with apoptotic MNC-secretomes.

Mean neuroscores (±SD) are plotted over time. Treated animals (red triangles for setting 1, Figure 5a, and red squares for setting 2, Figure 5b) improved over time compared to controls (black/white triangles for setting 1, Figure 5a, and black/white squares for setting 2, Figure 5b). Error bars correspond to +/- one standard deviation.

Apoptotic MNC-secretomes activate signaling cascades involved in cytoprotection in glia cells

Glia cells are non-neuronal cells that provide support and protection for neurons in the brain and peripheral nervous system (Giaume et al., 2010). We therefore investigated the potency of hMNC^{apo sec} to activate/phosphorylate signaling-molecules that are part of protective pathways in human primary astrocytes as well as in Schwann cells. For Western blot analysis, astrocytes and Schwann cells were treated with hMNC^{apo sec} for 1 hour. Both cell types showed an increased phosphorylation of CREB, Erk1/2, c-Jun, and Akt. Phosphorylation of HSP27 was only detected in astrocytes (Figure 6).

Figure 6. Expression of cytoprotective proteins in human Astrocytes (AC) and Schwann Cells (SCs).

Cell extracts were prepared after stimulation with hMNC^{apo sec} or cell culture Medium as control. Western blot analysis revealed an activation of CREB, ERK1/2, c-Jun, Akt, and HSP27. Proteins were normalized to the respective non-phosphorylated proteins.

Apoptotic MNC-secretomes induce CREB phosphorylation and neuronal sprouting in human primary neurons and contain BDNF

We next investigated whether hMNC^{apo sec} are also effective in human primary neuron cultures. Western blot analysis of neurons and astrocytes revealed a rapid dose dependent activation of CREB phosphorylation (Figure 7a). Incubation of neurons with apoptotic MNC-secretomes led to a significant increase in the length of newly sprouting neurons. Cultured neurons treated with hMNC^{apo sec} had a mean neurite length of 21±1µm (mean±SEM) versus 13±1µm in neurons treated with control medium (t-test; * p<0.0001; Figure 7b–c).

Figure 7. Enhanced CREB phosphorylation and neurite length in neurons treated with apoptotic MNC-secretomes.

(a) Cell extracts were prepared after stimulation with hMNC^{apo sec} or cell culture medium as control. Western blot analysis for phospho-CREB revealed a dose dependent activation of CREB in astrocytes and in neurons. (b) Neuron cultures treated with hMNC^{apo sec} or control medium for five days were stained with methylene-blue. One representative picture of ten is shown. Bar=10 µm (c) Lengths of neurons treated with hMNC^{apo sec} or cell culture medium as control were calculated using ImageJ software. Bars represent the mean of five different cultures.

In order to characterize the composition of neurotrophic factors present in hMNC^{apo sec}, we performed ELISA for BDNF, GDNF and NGF. Interestingly, only high amounts of BDNF (356±14pg/mL) were detected in hMNC^{apo sec}, suggesting an exclusive role for this neurotrophic factor in hMNC^{apo sec} (Figure 8a).

Figure 8. Profile of neurotrophic factors in hMNC^{apo sec} and animals treated with hMNC^{apo sec}.

(a) ELISA for BDNF, GDNF, and NGF detected only levels of BDNF (356±14pg/mL, mean±SEM) in hMNC^{apo sec}. (b) Six animals received an intraperitoneal injection with hMNC^{apo sec} (n=3, red bar) or control medium (n=3, black/white bar) and BDNF plasma levels were determined 24 hours after administration using ELISA.

Effects of apoptotic MNC-secretomes on plasma BDNF-levels in rats

After revealing BDNF as one component in hMNC^{apo sec}, BDNF levels in plasma of rats treated with hMNC^{apo sec} were measured with ELISA 24 hours after i.p. administration. Plasma BDNF-levels were higher in rats treated with hMNC^{apo sec} compared to those treated with control medium (n=3 for each compound). Twenty four hours after treatment with hMNC^{apo sec}, BDNF plasma levels were 72±5ng/mL (mean±SEM) compared to undetectably low BDNF levels in controls (Figure 8b).