Edited by: Antonio Gambardella, Magna Græcia University, Italy
Reviewed by: Jennifer Larimore, Agnes Scott College, United States; Carmelo Sgobio, German Center for Neurodegenerative Diseases (HZ), Germany
*Correspondence: Ren-Zhi Zhan
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Although development of cognitive decline in cancer patients who receive chemotherapy is common, the underlying mechanism(s) remains to be identified. As abnormalities in adult hippocampal neurogenesis may serve as substrate for cognitive dysfunction, the present study examines the effect of cyclophosphamide (CPP), a widely prescribed chemotherapeutic agent, on dendritic development of adult-born hippocampal granule cells in the rat. CPP was intraperitoneally injected into male Sprague-Dawley rats once a week for four consecutive weeks. Four weeks and 1 week after the last dose of CPP, Morris water maze test and doublecortin (DCX) immunohistochemistry were carried out to determine the effects of CPP on cognitive function and the rate of hippocampal neurogenesis, respectively. Adult newborn hippocampal granule cells were labeled at the same day as the first dose of CPP and were examined 10 weeks after labeling. Results showed that cognitive decline induced by CPP was associated with both suppressed adult hippocampal neurogenesis and abnormal development of dendrites of newborn granule cells. The abnormalities of dendrites in newborn granule cells after CPP exposure included less dendritic branching, shorter total dendritic length, thinner and torturous dendritic shafts with intermittent appearances of varicosities, and lower spine densities of stubby and thin types along dendritic shafts, but an increased density of mushroom-like spines. Adult-born granule cells in the presence of CPP, a widely used anti-cancer medication, display abnormal dendritic morphologies and fewer dendritic spines which may underlie cognitive dysfunction.
Systemic cancer treatment is associated with cognitive decline that manifests as impairments in learning, memory formation, attention, information processing speed and executive functioning (Anderson-Hanley et al.,
As a subcortical structure, the hippocampus functions in learning, memory formation and mood regulation. Granule cells in the hippocampal dentate gyrus are continuously to be generated throughout life in this brain region from neural stem/progenitor cells that reside in the subgranular zone. Ultimately, nearly a quarter of newborn granule cells survive to become mature neurons under normal conditions (Christian et al.,
All animal experiments were approved by the Animal Ethics Committee of Shandong University School of Medicine and performed according to the guides for the care and use of laboratory animals set by National Research Council (US). Male Sprague-Dawley rats (160–180 g, 6–8 weeks old) purchased from Beijing HFK Bioscience Company (Beijing, China) were housed in a university animal facility with a 12 h light/dark cycle and free access to food and water, and were weighed weekly before and during the experiments. In total, 34 animals were used in the present study; two animals died after CPP monohydrate (CPP) injection (all were in the 3rd week after initiation of injection). Three sets of experiments were conducted in 32 animals: 14 for Morris water maze testing, six for assessment of adult neurogenesis with doublecortin (DCX) immunohistochemistry and 12 for dendritic measurements following intradentate retroviral vector injection.
CPP monohydrate (Sigma, St. Louis, MO, USA) was dissolved in sterile saline to make a 50 mg/ml of stock solution. The stock solution was stored at −80°C until use. CPP was intraperitoneally injected into animals once a week for four consecutive weeks at either 25 mg/kg or 50 mg/kg. Animals received equivalent amount of saline in a schedule same to CPP-treated ones were used as controls.
Each animal was deeply anesthetized with an intraperitoneal injection of sodium pentobarbital (80 mg/kg). After complete paralysis, the animal was perfused through the aorta with 50 ml heparinized saline, followed by 300 ml of 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 6.8) over a period of 40 min. The brain was removed from the skull upon completion of perfusion, and then post-fixed in the same fixative at 4°C overnight. Thereafter, the brain was sequentially immersed in 10% sucrose in 0.1 M PBS for 4 h, 15% sucrose in 0.1 M PBS for 8 h, and finally 20% sucrose in 0.1 M PBS at 4°C overnight. Tissue block including the hippocampus was made with the aid of an appropriate brain matrix (WPI, Sarosata, FI, USA). After the tissue block was firmly embedded with a medium that consisted of 30% (w/v) chicken egg albumin (Sigma, St. Louis, MO, USA), 0.5% (w/v) gelatin, and 0.9% (v/v) glutaraldehyde in 0.1 M PBS as described previously (Zhan and Nadler,
The effect of CPP on the rate of neurogenesis was compared in saline- and CPP-injected rats (three animals each group) by staining and counting DCX-positive newborn granule cells. Brain sections were prepared 1 week after the final dose of CPP. Sections (40 μm in thickness) localized between 3.0 mm and 3.6 mm posterior to the bregma were serially cut. Sections with the order of number 3, 9 and 15 were subjected to DCX immunohistochemical staining with the free-floating procedure. After the collected sections were washed with 0.1 M PBS, they were incubated in a solution that consisted of 30% methanol and 3% H2O2 in water for 30 min to inactivate endogenous peroxidase. After rinsing, sections were incubated in a blocking solution that consisted of 2.5% BSA (Sigma, St. Louis, MO, USA), 0.2% Triton X-100 and 5% normal donkey serum in 0.1 M PBS at 4°C for 2.5 h to minimize non-specific reactions. Without rinsing, the sections were then incubated with a goat anti-DCX (Santa Cruz, San Jose, CA, USA) that was diluted with the blocking solution (1:400) at 4°C overnight. After three washes with 0.1 M PBS, the sections were incubated with biotinylated donkey anti-goat IgG (Jackson ImmunoResearch, West Grove, PA, USA) in a dilution of 1:400 at 4°C for 1.5 h. After rinsing with 0.1 M PBS, the sections were incubated with the avidin-peroxidase complex (Vector Labs., Burlingame, CA, USA) at 4°C for 60 min. After several washes with 0.1 M PBS, immunoreactions were visualized by an incubation with 3,3-diaminobenzidine, enhanced by the addition of 0.08% ammonium nickel sulfate (Vector Labs., Burlingame, CA, USA). The reaction was terminated by incubating the section with water. Sections were then flatly mounted on slide glasses and left to be air-dried for at least an hour. After dehydration with a series of graded ethanol solutions, the sections were then cleared in xylene and coverslipped with neutral balsam. DCX-positive cells in the dentate gyrus were counted and the lengths of the superior and inferior pyramidal blade of the dentate gyrus were measured in each section by using Image-Pro-Plus (Media Cybernetics, Rockville, MD, USA). The density of DCX-positive cells was calculated by dividing the number of cells with the total length of granule cell layer and expressed as the number of cells/mm. Values in three sections each animal were averaged.
For animals to be used for behavioral studies, open field test assessing general locomotor activity was conducted to qualify animals before CPP exposure. To this aim, a 90 cm × 90 cm white board, divided into 36 equal squares by red lines was used as a field apparatus. Each side of the board was fenced to prevent animals to jump out. Animals were individually placed in the central square and allowed to explore the area freely. The numbers of crossing (squares crossed), rearing (standing on hind limbs), grooming (washing face) and urination were recorded for 5 min. After each trial, the apparatus was cleaned with ethanol. Qualified rats were randomly divided into two groups, receiving intraperitoneal injection of saline (control group) or CPP (CPP group); both were carried out once a week for a consecutive 4 weeks. Four weeks after the last injection, the Morris water maze was conducted to assess animals’ spatial learning and memory abilities. The water maze task was carried out for seven consecutive days in which the first 6 days were used to train the animals to find the hidden platform, whereas the last day was used to probe how well the animals remembered the position of the platform. A cylindrical pool (120 cm in diameter), filled with water (22–25°C) was placed in a quiet testing room. The cylindrical pool was equally divided into four quadrants with a removable platform (10 cm in diameter) to be located in the center of the first quadrant. The platform was set 2 cm below the surface of water to make the platform invisible to animals. A digital camera was mounted above the pool and linked to an automatic tracking system (SMART polyvalent video-tracking system, Panlab, Spain) to track and record the performance of individual animals. Several bright-colored cues posted on the wall of the maze were used to help animals to find the platform. In the training sessions, each rat was put into the pool from the four different quadrants with its head facing the wall to prevent the animal seeing the platform directly. If a rat could find the platform within 60 s, it would be allowed to stay onto the platform for 20 s before removal from the pool. Instead, if a rat failed to find the platform within 60 s, it would be guided onto the platform. The time taken to find the hidden platform (latency to escape) was recorded. On the 7th day, the platform was removed and each rat was put into the pool from a start point (the opposite quadrant of the first quadrant) to probe its memory. The rat was allowed to swim freely in the pool for 60 s and the path of navigation was traced. Swimming speed and the time spent in the target quadrant and non-target quadrants were collected.
Concentrated CAG–GFP retroviral vector was made from an established stable virus-producing platinum-E cell line (Gao et al.,
After an animal was anesthetized with an intraperitoneal injection of sodium pentobarbital (25 mg/kg) and ketamine (60 mg/kg), the animal was placed on a stereotaxic frame suitable for rats (Stoelting, Wood Dale, IL, USA). After the skin was sterilized with a sequential application of 7.5% povidone iodine and 75% alcohol, the scalp was incised and a skull hole on the right side was drilled. One microliter of CAG–GFP retroviral vector was injected into the right dentate gyrus at a rate of 0.1 μl/min using a 1 μl Hamilton syringe (Bonaduz, GR, Switzerland) that was connected to an automatic injection pump (Stoelting, Wood Dale, IL, USA). The injection site was located 3.2 mm posterior from the bregma and 2.5 mm right to the midline, in a depth of 2.4 mm (measured from the surface of the cortex). The needle was left in the place for additional 15 min after the completion of injection to prevent possible backflow before removal. All surgical procedures were performed under sterile conditions. Animals were returned to cages after recovery from anesthesia.
For dendritic measurements, transcardial perfusion was performed 10 weeks after retroviral vector injection. Coronally cut hippocampal sections (110 μm in thickness) in which GFP-labeled granule cell(s) presented were subjected to GFP immunostaining by the free-floating procedure. Sections were first washed with 1XPBS, and then incubated in a blocking solution that consisted of 2.5% BSA, 0.2% Triton X-100 and 5% goat serum in 1XPBS to minimize non-specific reactions. Thereafter, sections were incubated with a mouse anti-GFP (Invitrogen, Carlsbad, CA, USA) in a dilution of 1:1000 at 4°C overnight. After three washes with 1XPBS, the sections were incubated with an Alexa fluor 488-conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA) in a dilution of 1:600. After another three washes with 1XPBS, sections were mounted on glass slides with 75% glycerol in PBS and coverslipped. Sections in which GFP was immunofluorescently amplified were scanned with the Zeiss 780 laser scanning microscope (Carl Zeiss, Jena, Germany). Images yielded were used for quantitative measurements of dendrite length, the complexity of dendritic arborization and dendrite spine.
To measure dendrite length and the complexity of dendritic arborization, individual GFP-labeled cells residing in the suprapyramidal blade were z-stacked under a 20× objective with a z-series interval of 1 μm and zoom 1. A 3-dimensional image created from the z-series file each cell was compressed into a 2-demnesional image. Only cells with relatively intact dendritic arborizations were quantitatively analyzed further. After GFP-positive soma and dendritic arborization were manually traced under ImageJ
z-stacks for dendritic segments located in the middle molecular layer were carried by using a 63× oil objective. The other parameters set for z-stacks were as follows: 0.12 μm of z-series thickness, zoom 4, and a spatial resolution of 0.03 μm × 0.03 μm × 0.12 μm. A 3-dimensional image was reconstructed from individual z-stack file. Spines were classified into stubby, thin or mushroom type based on spine length, head diameter to neck width ratio, and the size of head specific to dentate granule cells (Tyler and Pozzo-Miller,
Data are expressed as mean ± standard error of the mean (SEM). Two-group data are compared with unpaired Student’s
To observe the effect of CPP on cognitive function, a dose of 50 mg/kg reported by a previous study (Christie et al.,
Reduced learning and memory abilities after cyclophosphamide (CPP) treatment revealed by Morris water maze test. Animals were tested 4 weeks after the last dose of CPP (50 mg/kg). Error bars are standard error of the mean (SEM).
To determine if cognitive dysfunction induced by CPP is associated with a slower rate of adult hippocampal neurogenesis, we performed DCX immunohistochemistry. In addition to 50 mg/kg of CPP, a smaller dose was tested also. As shown in Figure
CPP reduces doublecortin (DCX)-positive cells in the hippocampal dentate gyrus.
At 10 weeks after retroviral vector injection, GFP-labeled cells from control animals were found to reside in the border between the granule cell layer and the hilus with apical dendrites extending throughout the molecular layer and tips ending at the hippocampal fissure (Figure
Representative images and statistical comparisons show the number of branch, total dendrite length and the complexity of arborization in the absence (Control) and presence of CPP. The first dose of CPP (50 mg/kg) was given at the same day as the CAG-GFP retroviral vector labeling and was continued for consecutive 4 weeks. Animals in the control group received equivalent amounts of saline. Animals in both groups were sacrificed 10 weeks after the injection of the retroviral vector.
After revealing abnormalities in the dendritic tree of newborn granule cells after CPP treatment, we then quantified spines in dendritic segments of the middle molecular layer. In the control group, dense protrusions in variable shapes appeared along the relatively regular dendritic shaft (Figure
CPP reduces total but increases mushroom-like dendritic spines in adult-born granule cells.
In addition to confirming that persistent cognitive decline induced by CPP is associated with a suppression of adult hippocampal neurogenesis, we found that CPP treatment leads to remarkable abnormal development of dendrites in newborn granule cells.
Multiple clinical surveys have provided solid evidence to show that chemotherapy can lead to cognitive decline, although the underlying mechanism remains to be fully elucidated (Anderson-Hanley et al.,
Clinical dosages of CPP vary according to the regimens of treatment. For intravenous injection, 40–50 mg/kg of CPP has been recommended for a consecutive 4 days (Cyclophosphamide FDA Packet Insert,
The Morris Water Maze was an established method for testing hippocampus-dependent learning, acquisition of spatial memory and long-term spatial memory. The substrate for those hippocampus-dependent cognitive deficits during and after CPP treatment has been firmly established. A recent study revealed that CPP treatment was associated with abnormal dendritic structures in hippocampal principal cells (Acharya et al.,
The development of dendrites in newborn granule cells is under strong regulatory control of the internal and external environments (Aimone et al.,
In conclusion, adult-born granule cells in the presence of CPP, a widely used anti-cancer medication, display abnormal dendritic morphologies and fewer dendritic spines, which may relate to chemotherapeutic agents-induced cognitive dysfunction.
LW did most of animal experiments, behavioral studies and imaging data processing. DG and QL did immunohistochemistry and data analysis. FG, XS and XW did confocal imaging. FW helped in experimental design. R-ZZ designed the study and wrote the article.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This study was supported by a grant from the National Natural Science Foundation of China (No: 31371183) to R-ZZ. The authors would like to thank Prof. Yanxun Liu and Mrs. Qian Zhu in the Shandong University School of Public Health for their help in statistical analyses.
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