Laminin degradation by matrix metalloproteinase 9 promotes ketamine‐induced neuronal apoptosis in the early developing rat retina

Abstract Aims During early development, laminin degradation contributes to the death of neurons. This study aims to investigate the role and regulation of laminin in ketamine‐induced apoptosis. Methods We performed terminal deoxynucleotidyl transferase biotin‐dUTP nick end labeling (TUNEL) and immunohistochemical assays to investigate the roles of the non‐integrin laminin receptor, matrix metalloproteinase 9 (MMP9) in ketamine‐induced neuronal apoptosis. In situ zymography, Western blot, and immunofluorescence were used to explore the relationships between laminin, MMP9 activity, and Zn2+. Experiments were performed using whole‐mount retinas dissected from Sprague Dawley rats. Results The TUNEL and immunohistochemical assays indicated that ketamine‐induced neuronal apoptosis in early developing rat retina. Blockade of non‐integrin laminin receptor promoted ketamine‐induced apoptosis, while non‐integrin laminin receptor activation attenuated ketamine‐induced apoptosis. Ketamine‐induced laminin degradation, possibly by enhancing the activity of MMP9. MMP9 inhibition reduced ketamine‐induced apoptosis by reducing laminin degradation. Downregulation of Zn2+ attenuated the increased MMP9 activity, laminin degradation caused by ketamine and significantly reduced ketamine‐induced neuronal apoptosis. Conclusion Laminin degradation by MMP9 promoted ketamine‐induced neuronal apoptosis in early developing rat retina. The non‐integrin laminin receptor may be a pathway involved in ketamine‐induced apoptosis. Zn2+ downregulation may play a protective role against ketamine‐induced neuronal apoptosis through inhibiting MMP9 activity.


| INTRODUC TI ON
Although the risk of learning and memory impairments associated with general anesthesia in children is still under debate, parents and the general public are very concerned about the safety and long-term outcome of children undergoing general anesthesia. 1,2 Furthermore, various animal studies have demonstrated that long-term or repeated exposure to general anesthetics can cause neuronal apoptosis in early stages of life and learning and memory impairments later in life. [3][4][5] General anesthesia-induced neurotoxicity in animals mainly occurs during the period of peak synaptogenesis, referred to as the window of vulnerability. 6,7 Although extensive studies have been carried out for the past 20 years, the mechanisms underlying general anesthesia-induced developmental neurotoxicity still need to be elucidated, and no effective prevention or treatment strategies have been developed thus far. 7,8 General anesthetics promote central nervous system inhibition via activation of the gamma-aminobutyric acid type A receptor (GABAA-R) and/or blockade of the N-methyl-D-aspartic acid receptor (NMDAR). 9 During early neuronal development, both GABAA-R activation and long-term NMDAR blockade can cause intracellular calcium disturbances and neuronal apoptosis. 8,10 In addition to these effects caused by the receptors, general anesthesia may also induce neuronal apoptosis by influencing the extracellular environment including brain-derived neurotrophic factor, inflammatory mediators, cell-extracellular matrix (ECM), etc [11][12][13] During early postnatal development, the ECM plays crucial roles in proliferation, migration, and differentiation of neural cells and form loose ECM fiber-like structures, which occurs in parallel with synaptic development. 14 The ECM consists of proteins including collagen, fibronectin, and laminin. 14 Laminins are indispensable building blocks of cellular networks and of extracellular polymers, which determine the architecture and physiology of basement membranes. 15 Previous studies have demonstrated that laminin degradation can lead to the death of developing neurons by affecting events downstream of protein kinase B (Akt) activation. 16 Plasmin-mediated laminin degradation is critical for ethanol-induced neuronal apoptosis. 17 These studies indicate that laminin may be a pathway leading to the apoptosis of developing neurons. However, whether laminin degradation contributes to the neuronal apoptosis induced by ketamine, a NMDAR antagonist, remains to be elucidated.
The effects of laminins are often mediated through interactions with integrin and non-integrin laminin receptors (LR). 18 The non-integrin LR was the first identified laminin receptor. 19 Interactions between the non-integrin LR and laminin play a key role in mediating changes in ECM that affect cell adhesion, neurite outgrowth, angiogenesis, and apoptosis. 20,21 The non-integrin LR is required for maintenance of cell viability by preventing apoptosis. 22,23 Previous study demonstrated that siRNA-mediated knockdown of non-integrin LR reduced FAK phosphorylation, leading to cell apoptosis. 24 However, whether non-integrin LR involved in ketamine-induced neuronal apoptosis needs to be elucidated.
Laminin can be targeted and proteolytically cleaved to regulate neuronal function by active matrix metalloproteinases (MMPs), which widely exist in the early developmental period. [25][26][27] A previous study demonstrated that MMP9 can regulate neuronal survival by degrading laminin and modulating the laminin-integrin β1 signaling pathway. 16,28 Moreover, as a zinc-dependent endopeptidase, the activity of MMP9 is closely related to the concentration of free zinc ions. Zn 2+ downregulation has been demonstrated to contribute to the inhibition of MMP activity. 29,30 Nevertheless, it remains unclear whether altering MMP9 activity by up-or downregulating Zn 2+ affects ketamine-induced neuronal apoptosis in early developing rat. We therefore speculated that altering MMP9 activity by up-or downregulating Zn 2+ may affect laminin degradation and ketamine-induced neuronal apoptosis. In the present study, we explored the possible effects of laminin and MMP9 on ketamine-induced neuronal apoptosis in the developing rat retina.

| Animals
All experimental procedures were approved by the Animal Care All rat pups were housed with their mothers under a 12-hour light/ dark cycle.

| Tissue dissection
Retinal tissues were prepared as previously described. 6 Briefly, the eyeballs of P7rats were rapidly removed with scissors following instantaneous decapitation. The extracted eyeballs were then further dissected in an ice-cold bath of artificial cerebrospinal fluid (ACSF) composed of the following (in mmol/L): 119 NaCl, 26.2 NaHCO 3 , 2.5 KCl, 1.3 MgCl 2 , 11 D-glucose, 1.0 KH 2 PO 4 and 2.5 CaCl 2 equilibrated with 95% O 2 and 5% CO 2 . To facilitate full exposure of the retina to drugs, an incision spanning approximately 1/5th of the circumference of the eyeball was made between the edges of the cornea and sclera. Following 1 hour of recovery in ACSF bubbled with a mixture of 95% O 2 /5% CO 2 gas at 37°C, the eyeballs were incubated with ketamine, antagonists or agonists (either in combination or separately) in ACSF bubbled with a mixture of 95% O 2 /5% CO 2 gas at 37°C for 5 hours. agonist Laminin (925-933) (Adooq Bioscience Company) and the LR antagonist NSC47924 (Yifei Biotechnology Company). All drugs were dissolved in ACSF except for NSC47924 and TPEN, which were first dissolved as a stock solution in DMSO, and then diluted to ACSF with a DMSO concentration <0.1%.

| Immunohistochemistry
Immunohistochemistry was performed according to experimental methods described previously. 6,9 After drug treatment, the retinas were dissected from the eyeballs in an ice-cold bath of ACSF and fixed in 4% paraformaldehyde for 24 hours. The fixed retinas were then incubated with ethanol and xylene, after which they were infiltrated with paraffin. The paraffin-embedded retinas were cut into 4-6 μm-thick slices using a microtome (Leica-2135, Leica). After endogenous peroxidase inactivation and heat-induced antigen retrieval, the tissue sections were first incubated with a primary antibody against cleaved caspase-3 (AC3; Table 1) overnight at 4℃. The sections were then incubated with a horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (IgG) secondary antibody (PV-9001, ZSGB-BIO) at 37°C for 1 hour.
For immunofluorescence, the retinal sections were first incubated with a primary antibody against laminin or MMP9 (Table 1) overnight at 4°C. They were then incubated with an Alexa Fluor 594-conjugated goat anti-rabbit IgG secondary antibody (Thermo Scientific) for 30 minutes, followed by DAPI for 5 minutes. Images were captured using a fluorescence microscope (Leica TCS SP8; Leica). Each image was composed of 1384 × 1040 pixels and had a resolution of 150 pixels/inch. Each group comprised five retina samples. Five discontinuous images randomly obtained using a fluorescence microscope were analyzed in each sample. To compare intensities between samples, the same exposure time was used for all samples. Image Pro Plus 6.0 (Media Cybernetics Company) was used to determine fluorescence intensity.

| Western blot assay
The protein concentration of retinal extracts and cell lysates was determined using a BCA kit (Beyotime Biotechnology), and 40 μg of proteins was electrophoresed in 10% SDS-PAGE. The separated proteins were then transferred to nitrocellulose membranes (Millipore).
Bovine serum albumin in Tris buffer saline was used to block nonspecific binding. The membranes were incubated with a primary antibody against MMP9 at 4°C overnight. GAPDH was used as a loading control ( Table 1). The membranes were then incubated with a horseradish peroxidase-streptavidin-conjugated secondary antibody for 1 hour at room temperature. Antibody detection was performed via enhanced chemiluminescence (Thermo Fisher Scientific), and the intensity of the bands was quantified by densitometric analysis using Gel Pro Analyzer software (Media Cybernetics, Inc).

| In situ gelatin substrate zymography
Fluorescent in situ gelatin substrate zymography was used to localize MMP9 proteolytic activity according to the manufacturer's

| Statistical analysis
Data are expressed as mean ± standard deviation. All statistical data were analyzed using GraphPad Prism 5 software (GraphPad Software Inc) or IBM SPSS Statistics 23 (SPSS Inc, IBM Corporation).
The Shapiro-Wilk test was used to assess the normality of the data distribution. Student's t test was used to analyze comparisons of normally distributed data. Multiple comparisons were performed using one-way analysis of variance followed by the least significant difference post hoc test. Data that do not exhibit a normal distribution were analyzed using the Mann-Whitney or Kruskal-Wallis test.
P values < .05 were considered statistically significant.

| Ketamine decreased the expression of laminin and MMP9 inhibition attenuated ketamineinduced decrease in laminin expression
To investigate the effects of ketamine on laminin expression, we

| Ketamine increased the expression and activity of MMP9 in developing rat retina
To detect potential changes in MMP9 after ketamine administration, the protein expression of MMP9 in the rat retina was examined by Western blot and immunofluorescence. Notably, 150 μmol/L ketamine significantly increased the expression of MMP9 in the rat retina ( Figure 4A,D, Supplementary Figure). Our immunofluorescence experiments revealed that exposure to ketamine increased the expression of MMP9 in the GCL of the retina at P7 (Figure 4B,E). A marked upregulation of MMP9 activity in the GCL of the retina was detected in rat pups after ketamine treatment using in situ zymography ( Figure 4C,F).

| Downregulation of Zn 2+ reduced the activity of MMP9 and laminin degradation in developing rat retina
To determine whether changes in the concentration of Zn 2+ affect MMP9 activity, we performed in situ gelatin substrate zymography in the rat retina. We found that 100 μmol/L ZnCl 2 significantly increased the gelatinolytic activity of MMP9 in the GCL of the retina.
Conversely, 100 μmol/L TPEN reduced MMP9 activity. TPEN also significantly attenuated the ketamine-induced increase in MMP9 activity in the GCL of the retina ( Figure 5A). The Western blot results showed that compared to the control group, the expression of cleaved MMP9 was increased by ZnCl 2 treatment and decreased by 100 μmol/L TPEN ( Figure 5B,C, Supplementary Figure).
We further explored the influence of Zn 2+ concentration on laminin expression in the GCL in P7 rats. The immunofluorescence experiments revealed that 100 μmol/L ZnCl 2 significantly reduced the expression of laminin in the GCL (P < .05). Conversely, 100 μmol/L TPEN increased the expression of laminin compared to the control group (P < .05).
Downregulation of Zn 2+ by TPEN also significantly attenuated the ketamine-induced decrease in laminin expression ( Figure 5D,E).

| Downregulation of Zn 2+ reduces ketamineinduced neuronal apoptosis in early developing rat retina
The immunohistochemistry and TUNEL assays revealed that exposure

| D ISCUSS I ON
Our study demonstrated that long-term ketamine exposure was able to induce neuronal apoptosis in early developing rat retina.
Blockade of the non-integrin laminin receptor promoted ketamineinduced neuronal apoptosis, while non-integrin laminin receptor activation attenuated ketamine-induced apoptotic responses.
Ketamine-induced laminin degradation, possibly through enhancing the activity of MMP9. Inhibition of MMP9 activity ameliorated ketamine-induced neuronal apoptosis through reducing laminin degradation. Furthermore, we also found that downregulation of Zn 2+ played a protective role against ketamine-induced neuronal apoptosis through inhibiting MMP9 activity and laminin degradation.
A previous study suggested that laminin degradation induced neuronal apoptosis in the newborn hippocampus. 16  pathway. The non-integrin LR plays important roles in cell migration, invasion, angiogenesis, ECM remodeling, and apoptosis. 22,32,33 However, laminin-integrin binding has also been reported to modulate neuronal survival through the Akt or focal adhesion kinase signaling pathway. 16,34 Whether the laminin-integrin signaling pathway is involved in ketamine-induced apoptosis needs further investigation.
Previous studies have demonstrated that laminin can be degraded by MMPs, including MMP9, during brain development. 16,28 Our study found that the MMP9 inhibitor increased laminin expression in developing neuron, which is consistent with the previous finding.  45 In addition, Hwang et al reported that upregulation of Zn 2+ increased the activity of MMP9 in the mouse brain. 46 Furthermore, our research found that upregulation of Zn 2+ aggravated ketamine-induced laminin degradation, and TPEN reduced the laminin degradation. The protective mechanism of TPEN in neuronal apoptosis may be related to that the decreased MMP9 activity leads to reduced degradation of laminin. Thus, it may be possible to reduce neurotoxicity caused by general anesthesia in children by modulating Zn 2+ to change the activity of MMP9 and laminin expression.
In the present study, the newborn rat retinas were used to examine the potential roles of zinc and MMP9 in neuronal apoptosis during early postnatal development. As an extension of the central nervous system, the retina is composed of three neuronal layers and provides an excellent model for the assessment of neuronal degeneration. 6 Furthermore, the use of newborn rat retinas overcomes limitations associated with in vivo animal experiments, namely the effects of hypoxia and CO 2 retention due to general anesthesia and physiological consequences of stress/physiological responses to stress.
With respect to the limitations of this study, we focused on cell counts to the GCL, but there are Caspase 3 + and TUNEL + cells present in places other than the GCL. The concentration of Zn 2+ in the retina was not detected after the administration of ZnCl 2 and TPEN. The mechanism that ketamine increased MMP9 expression and activity in early development needs further study. Furthermore, we did not study the role of integrin in laminin degradation-induced apoptosis. Therefore, the roles of LRs and other signaling pathways in ketamine-induced neuronal apoptosis during early development require further exploration.

| CON CLUS ION
In conclusion, this study showed that laminin degradation by MMP9 promoted ketamine-induced neuronal apoptosis in early developing rat retina. The non-integrin LR may be a pathway involved in ketamine-induced apoptosis. Zn 2+ downregulation may play a protective role against ketamine-induced neuronal apoptosis through inhibiting MMP9 activation and laminin degradation.

ACK N OWLED G M ENTS
This study was supported by The National Natural Science Foundation of China (Beijing, China; grant no. 81771218 to Jijian Zheng)

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.