Description

INTRODUCTION

Gabapentin was originally approved to treat neuropathic pain, seizures, and postherpetic neuralgia [1]. Research suggests that its mechanism of action may involve the increased synthesis of gamma-aminobutyric acid (GABA) and the inhibition of glutamate neurotransmitter pathways, which affect neural cells [2,3]. Although gabapentin is categorized as a GABA analog, it is crucial to note that it does not directly interact with GABA receptors. Instead, its primary function is to inhibit voltage-gated calcium channels, particularly the α2δ-1 subunit [2,3,4,5]. This inhibition is vital for regulating neurotransmitter release, which in turn reduces neuronal excitability and synaptic transmission.

Interestingly, gabapentin has been shown to decrease reactive gliosis [6], an inflammatory response in the central nervous system (CNS) that can lead to further damage to neural cells. Between 2009 and 2016, the rate of gabapentin prescriptions nearly doubled across the United States, with increases ranging from 44% to an astonishing 179% in various states [1,7]. By 2019, gabapentin had risen to the seventh most prescribed medication in the country, with an impressive 69 million prescriptions written that year [7]. While gabapentin is generally deemed safe when used alone, combining it with opioids or benzodiazepines raises significant concerns due to the heightened risk of overdose. This trend underscores the potential dangers associated with the increasing use of gabapentin.

Recent research, including a thorough analysis by the CDC, has highlighted the serious consequences of gabapentin use. A review of 62,652 overdose deaths from 2019 to 2020 across 24 jurisdictions revealed that gabapentin was detected in 5,687 cases (9.7%) among the 58,362 deaths with toxicology results [1]. Alarmingly, gabapentin was implicated in overdose deaths for 2,975 individuals, representing 52.3% of those who tested positive for the drug [8,9]. The demographic data indicated that a significant majority of these overdose deaths involved non-Hispanic White individuals (83.2%) and those aged 35 to 54 years (52.2%) [1,7].

These statistics highlight a pressing concern: over half of the overdose fatalities among individuals with a positive gabapentin toxicology test are directly linked to the drug. Despite its widespread use, the mechanisms behind gabapentin toxicity, overdose, and mortality remain inadequately understood. This knowledge gap emphasizes the need for further research to clarify the risks associated with gabapentin, particularly when used alongside other central nervous system depressants.

As healthcare professionals navigate the complexities of gabapentin prescribing, it is vital to weigh its therapeutic advantages against potential risks. Raising awareness and providing education about the dangers of gabapentin, especially in combination with other medications, is essential for reducing associated risks. The surge in gabapentin prescriptions serves as a reminder of the importance of careful prescribing practices and ongoing research to ensure patient safety in a changing medical environment.

Gabapentin has become a fundamental treatment option for various neurological disorders, providing relief to many patients suffering from conditions such as neuropathic pain, seizures, and postherpetic neuralgia. Its applications extend beyond these primary uses, as it is often prescribed off-label for a variety of other conditions, including Restless Leg Syndrome (RLS), insomnia, diabetic neuropathy, tremors, cocaine withdrawal, cancer-related hot flashes, and even amyotrophic lateral sclerosis (ALS) [1,2,3,4,5]..

However, the response of human astrocyte cells to increasing concentrations of gabapentin in molar ratios has yet to be thoroughly investigated, even as its clinical use continues to rise. In a series of studies, human astrocytes were exposed to various molar ratios of gabapentin to explore their responses, providing deeper insights into the biomolecular mechanisms of the drug.

2.0 METHODOLOGY

2.1 The methods used in the groundbreaking American Academy of Primary Care Research (AAPCR) human astrocyte study has also been published by Okea RN and Osuji GO [8,9].

2.2 Preparation of Astrocyte Medium and Gabapentin Concentrations

The methods, which have been detailed in recent scientific publications [9], involve the preparation of Astrocyte Medium and the administration of gabapentin concentrations. The Astrocyte Medium was prepared according to a protocol from iXcell, where key components—Fetal Bovine Serum (FBS), Growth Supplement, and Antibiotic-Antimycotic—were thawed in a 37°C water bath and mixed aseptically in a Biological Safety Cabinet (BSC). Specifically, the preparation included 50 mL of FBS, 1 mL of Growth Supplement, and 5 mL of Antibiotic-Antimycotic added to 500 mL of iXCell HA medium, resulting in final concentrations of 10% FBS, 0.2% Astrocyte Supplement, and 1% Anti-Anti [10,11,12,13,14,15,16,17].

To create drug-infused med ia formulations, non-sterile drugs were sterilized by filtration through a 0.22 μm sterile filter within the BSC to prevent contamination during cell culture. This meticulous approach ensures the integrity and sterility of the experimental conditions.

2.3 Preparation of Molar Ratios of Gabapentin Media Solutions

The preparation of Gabapentin Media Solutions involved creating various concentrations of gabapentin. For the 100 mM solution, 3009 mg of gabapentin was dissolved in 177 mL of complete Astrocyte Medium and filtered through a 0.22 μm sterile filter. The same procedure was applied for the 50 mM concentration, using 1800 mg of gabapentin in 211.76 mL of medium, and for the 25 mM concentration, with 900 mg of gabapentin in 209.3 mL of medium.

For the 5 mM solution, 300 mg of gabapentin was initially dissolved in 7.97 mL to create a 220 mM concentration. Then, 4.55 mL of this 220 mM solution was mixed with 195.45 mL of medium to achieve the final 5 mM concentration. Each solution was also filtered through a 0.22 μm sterile filter to ensure sterility [11,12].

Cell numbers and morphology were documented as done by Okea RN and Osuji GO [8,9].

3.0 RESULTS

The results from this study is broadly divided into cell indices and the culture image slides.

3.1 Cell Indices

In the control group A, human astrocyte cultures exhibited remarkable growth, expanding from 0.375 million cells at the time of seeding to an impressive 8.71 million cells by day 9, following a sigmoidal and exponential growth pattern. In contrast, the 25 mM gabapentin group A only increased to 0.629 million cells from the initial 0.375 million, demonstrating a linear growth pattern that disrupted the sigmoidal and exponential trends observed in the control groups (see Figures 1, 2, 3, and 4). By day 11, two days after reaching confluence, control group B experienced a significant cell loss of 1.92 million cells, while the 25 mM gabapentin group B showed a minimal attrition of just 40,000 cells during the same timeframe. These findings indicate that both growth and attrition rates were notably reduced in the 25 mM gabapentin groups compared to the control groups (refer to Figures 3 and 4).

Cell diameter continued to increase over time from the initiation of the culture, with a particularly pronounced growth in the 25 mM gabapentin groups (see Table 1). The increase in cell size followed an approximately linear trend within these groups. Additionally, the size increase demonstrated a dose-dependent relationship (see Table 2), comparing the control group with the 5 mM and 25 mM gabapentin groups. Notably, measurements taken on day 9 and day 11 illustrated that cell diameter increased in correlation with the gabapentin concentration.

Table 3 presents cell viability data, which showed no specific trends between the control and 25 mM gabapentin groups. Overall, both groups maintained a high level of cell viability, exceeding 95% at all time points.

Table 4 details the calculations for doubling time (DT) across the control, 5 mM gabapentin, and 25 mM gabapentin groups, revealing that DT increased with higher gabapentin concentrations. The control group had a DT of 1.98 days, while the 5 mM gabapentin group took 2.90 days, and the 25 mM gabapentin group required an extended 12.06 days for cell counts to double. Interestingly, the 25 mM gabapentin group is unlikely to achieve doubling within 12 days, as cell growth begins to decline after day 9, leading to a net loss of cells.

Table 6 outlines the mean lifetime (MLT) calculations for the control, 5 mM gabapentin, and 25 mM gabapentin groups. These values also reflect a dose-dependent trend, indicating that MLT increases with higher gabapentin concentrations. The estimated MLT for the control group was 218 hours, 309 hours for the 5 mM gabapentin group, and an impressive 755 hours for the 25 mM gabapentin group. These results are statistically significant (p < 0.004, p < 0.005, or p < 0.0008, respectively) – see Table 7.

                                      Figure 1                                                                                                      Figure 2

                                  Figure 3                                                                                                   Figure 4

Table 1: Human Astrocyte cell diameter on various days for the Control and 25 mM Gabapentin Groups 

Table 2: Human Astrocyte cell diameter on various days for the Control; 5mM Gabapentin and 25 mM Gabapentin Groups

Table 3: Human Astrocyte Cell Size for 5mM Gabapentin and 25 mM Gabapentin Groups

Table 4: Human Astrocyte cell viability on various days for the control and 25 mM Gabapentin Groups

Table 5: Calculation of the Doubling Time (DT) from Study Raw Data using the formula

(DT) = T Ln2 / Ln (N9/N1) 

Table 6: Calculation of the Mean Lifetime of Human Astrocyte Cells after the cells attained confluency.

Table 7: Calculation of Test of Significance for Mean Life time (MLT)

3.2 Cell Electron Microscope Images:

The second dataset comprises daily microscopy and photography performed under consistent conditions across all T-Flasks. Using a phase contrast electron microscope, we obtained images at two magnifications (4X and 10X) to provide a thorough visualization of the cell cultures. The 4X images, illustrated in Figures 5 to 8, offer a general overview of cell morphology and density, aiding in the assessment of the culture's overall health. Although the 10X images are not included here, they deliver a more detailed perspective of individual cells, enabling a closer inspection of their structural integrity and any potential irregularities.

These daily microscopy sessions served several functions, including evaluating morphology, monitoring cell viability, and inspecting for contamination. This approach allowed for direct visualization of cell shape and structure, observation of growth patterns, and assessment of the cultures' overall health, while also ensuring that no foreign organisms were present in the culture media, which could compromise the results [18]. The standardized microscopy records were vital for conducting numerical comparisons using ImageJ software, a tool designed for assessing cell density. This analysis not only bolsters the reliability of our findings but also establishes a solid framework for understanding the effects of gabapentin on cell culture behavior.

Consequently, the integration of quantitative data on cell count, size, and viability, along with qualitative microscopy observations, created a comprehensive dataset that supports our investigation into the effects of gabapentin on HAC cultures. The application of phase-contrast electron microscopy to examine cellular growth patterns has proven to be an invaluable technique, yielding significant insights into the dynamics of experimental cultures. The images captured (Figures 5 to 8) revealed remarkably similar growth patterns for both control groups A and B, as well as for gabapentin groups A and B, facilitating a meaningful side-by-side comparison across the culture flasks. This meticulous methodology not only reinforced our findings but also ensured both internal and external consistency [18,19,20,21].

Figures 5, 6, 7, and 8 are displayed below, showcasing real-time comparative phase contrast electron micrograph images of HAC for the control groups and the 25mM gabapentin groups as labeled.

                                                                                                                     Figure 5

                                                                                                                          Figure 6

                                                                                                                          Figure 7

                                                                                                                        Figure 8

4.0 DISCUSSION 

4.1 Reliability and Reproducibility

The experimental design discussed incorporates multiple layers of controls and pairs to enhance reproducibility, reliability, and validity of results while minimizing inter-observer variability. It emphasizes that experiments should not merely aim to fulfill statistical requirements, such as creating triplicates for centrality measures, if they do not address the scientific questions intended. In this study, a high intraclass correlation coefficient (>0.95) suggests that triplicates are unnecessary, especially when using similar cell lines under identical growth conditions [8,9,19]. The research uniquely combines cell culture and dose-response studies, allowing for robust internal and external controls that validate the experiment's reliability. This approach is both simple and cost-effective, reducing the need for numerous replicates while ensuring internal validity through an escalating dose-response model [20,21,22].

4.2 The Effects of Gabapentin on HAC Growth

This study investigated the effects of gabapentin on human astrocyte cells (HAC), revealing significant impacts on cell growth and viability. Gabapentin decreased the exponential growth rate of HAC before reaching confluence, with a notable reduction at a concentration of 5 mM compared to control groups. After reaching confluence on day 9, both the control group and the 5 mM gabapentin group experienced cell attrition, with the control group showing a 22% increase in apoptosis and a decline in cell numbers from 8.72 million to 6.79 million by day 11. The 5 mM gabapentin group also saw a decrease in HAC numbers, dropping from 3.22 million to 2.72 million (15% decline) [8]. In contrast, the 25 mM gabapentin group exhibited a reduced rate of cell loss post-confluence, with only a 6% drop in cell count from 629,000 to 590,000.

These findings indicate a dose-dependent inhibition of HAC growth by gabapentin before confluence, while post-confluence, a higher concentration appears to decrease the rate of apoptosis. The study suggests that gabapentin may influence cellular dynamics in neurobiological systems, warranting further research into the mechanisms behind these effects and their potential therapeutic implications.

4.3 The Effect of Gabapentin on the Time to Attain Confluency in HAC Cultures.

In a controlled study examining the effects of gabapentin on human astrocyte cells, research scientists established two distinct groups: a control group cultured without any pharmaceutical intervention and a gabapentin group treated with 25 mM gabapentin. By keeping all other conditions constant, the study aimed to isolate gabapentin's effects on astrocyte growth patterns.

From as early as day 2, the scientists noted observable differences in cell growth and density in the astrocyte cultures, as illustrated in the T-75 flask images (Figures 5 to 8). The 5 mM gabapentin groups displayed a slightly lower cell density compared to the control groups, a trend that persisted throughout the culture period and became more pronounced over time. This oligemic appearance in the gabapentin groups indicated that the 25 mM concentration of gabapentin effectively reduced the growth rate of astrocytes. However, the growth pattern remained sigmoidal, with both groups reaching confluency by day 9. This critical observation suggests that gabapentin did not influence the time to confluency in astrocyte cultures, challenging the long-held belief in cell biology that cell culture confluency depends solely on the available surface area.

Traditionally, cellular biology posits that adherent cells, such as human astrocytes, cease growth upon contact with themselves and with limiting culture surface area, a phenomenon known as contact inhibition [23, 24]. The fact that the gabapentin group reached confluence simultaneously with the control group raises questions about this long-standing dogma, suggesting that external agents like gabapentin can modulate cell counts at confluence and beyond without affecting the time to reach confluence. Logically, one would expect that a slowed cell growth rate and precocious aging would prolong the time for the culture to cover the plate’s surface area, but this was not the case. Similar results emerged with 5 mM gabapentin in the study by Okea RN and Osuji GO [8].

4.4 The Effect of Gabapentin on Cell Size

In cellular biology, cell size is often considered an indicator of aging, with cells typically increasing in size as they age [25,26]. This study observed a similar trend in human astrocyte cells. The control group showed a modest increase in cell size from 14.8 µm on day 1 to 15.7 µm by day 9 (6% increase), and further to 15.8 µm by day 11 (6.7% overall increase). In contrast, the 5 mM gabapentin group exhibited a more significant increase, reaching 16.6 µm by day 9 (12.2% increase) and 17.0 µm by day 11 (14.9% increase), with a statistically significant difference between the two groups (p = 0.033) [8].

The 25 mM gabapentin group showed even larger cell sizes, measuring 18.4 µm (24% increase) on day 9 and 19.6 µm (32% overall increase) on day 11, with a statistically significant difference between the two groups (p = 0.012) – see Table 2; suggesting that gabapentin may accelerate the aging process of astrocyte cells in a dose-dependent manner. Interestingly, while gabapentin appears to reduce growth rates and increase cell size, the fewer surviving cells in the treated groups exhibited longer lifespans, indicating a complex relationship between drug treatment, cell growth, and aging.

4.5 The Effect Gabapentin on Human Astrocyte Longevity

In the post-confluence phase, we quantified the decline in cell growth using the decay constant equation (Table 4). The control group exhibited a decay constant rate (λ) of 4,590 cells/hr, while the 5 mM gabapentin group showed a lower decay constant rate of 3,230 cells/hr [8]. For the 25 mM gabapentin group, we determined a decay constant (λ) of 1,320 cells/hr (see Table 6). This translates to a mean lifetime of 218 hours for the control group, compared to 309 hours for the 5 mM gabapentin group and 755 hours for the 25 mM gabapentin group—an increase of 91 hours and 537 hours in survival time, which was statistically significant (p = 0.004 and p = 0.005, respectively). These findings indicate that astrocyte cells exposed to 5 mM and 25 mM gabapentin after confluence were less likely to survive, but the surviving cells lived longer compared to their untreated counterparts (see Table 6).

Our findings suggest that gabapentin at concentrations of 5 mM and 25 mM may significantly impact the mean lifetime expectancy of human astrocyte cells (HACs). This concentration could enhance the resilience of these cells, allowing them to adapt to metabolic stress, or it may induce a level of stress that triggers a survival mechanism, ultimately delaying age-related programmed cell death in the fewer surviving cells. These intriguing hypotheses warrant further investigation, particularly through studies utilizing escalating concentrations of gabapentin. The adaptation of HACs to gabapentin exposure and toxicity may represent a critical biological process within the cells.

Moreover, the increased longevity of astrocytes in the presence of gabapentin raises important questions about the drug's role in cellular aging and its potential therapeutic implications. Future research should delve deeper into the mechanisms underlying these observations to better understand gabapentin's multifaceted effects on cellular biology.

4.6 Molar Ratios and Gabapentin Effects on HACs

This study reveals a critical insight: administering gabapentin in molar concentrations is essential for achieving consistent and predictable biological responses. This phenomenon aligns with the fundamental principle that molecular interactions in chemistry occur in molar ratios. The results clearly show that gabapentin at 5 mM and 25 mM significantly suppresses the growth of HACs, with this effect directly linked to molar concentrations. The predictable responses observed emphasize the necessity of measuring gabapentin concentrations in molar ratios. Without this crucial design component, cellular and chemical interactions lack clarity, highlighting the importance of molar ratios in both chemical and biological systems. This also suggests that cell-to-cell interactions in biological systems occur in molar ratios [8,25].

The clinical implications of these findings are profound. Currently, physicians prescribe gabapentin based on weight measures such as milligrams and grams, which may not yield the consistent and predictable responses observed at the cellular level in this study. Recognizing that chemical and molecular reactions occur in molar ratios is vital, especially in pharmacology and clinical medicine. This understanding could lead to more effective dosing strategies that align with the cellular responses we observed.

The suppression of HAC growth with increasing doses of gabapentin reflects the drug's toxicity pattern toward human astrocyte cells. Importantly, the gabapentin used in this research matches what physicians prescribe, underscoring the need for a deeper exploration of its toxicology in vivo, particularly in molar ratio concentrations. Given that gabapentin ranks among the top seven prescribed medications in the USA for various neurological conditions, millions of patients are exposed to this drug. Therefore, thoroughly understanding its therapeutic and toxicological profiles is essential to ensure patient safety and maximize therapeutic benefits.

These results indicate that gabapentin at 5 mM and 25 mM concentrations can lead to varying levels of HAC growth suppression and promote apoptosis; and cause surviving cells to live longer. This raises critical questions about the mechanisms underlying gabapentin's effects and addressing these questions through focused research could significantly enhance our understanding of gabapentin's pharmacology, therapeutic applications, and toxicity.

The findings from this study open new avenues for research into gabapentin's effects on human astrocyte cells. By investigating the drug's mechanisms of action and its implications for patient care, we can better navigate the complexities of its use in clinical settings, ensuring both safety and efficacy for those who rely on this medication.

5.0 CONCLUSION

These results indicate that gabapentin at 25 mM concentration and, in the previous study, at 5 mM concentrations, can lead to varying levels of HAC growth suppression and exacerbate cell apoptosis; but at the same time cause the surviving cells to live longer. This raises critical questions about the mechanisms underlying gabapentin's effects and addressing these questions through focused research could significantly enhance our understanding of gabapentin's pharmacology, therapeutic applications, and toxicity. The clinical implications of these findings are profound, physicians prescribe gabapentin based on weight measures, which may not yield the consistent and predictable responses observed at the cellular level in this study. Recognizing that chemical and molecular reactions occur in molar ratios is vital, especially in pharmacology and clinical medicine. This understanding could lead to more effective dosing strategies that align with the cellular responses we observed. This study also enriches our understanding that molar concentration formulations of gabapentin, and all drugs for that matter, are vital ingredients to elicit maximum cellular responses, and avoiding overdose and toxicity.

This study also raises questions about the long-standing dogma called contact inhibition in cell biology, suggesting that external agents like gabapentin can modulate cell counts at confluence and beyond without affecting the time to reach confluency, thereby modulating the mean lifetime survival of certain cells groups. All these observed effects of gabapentin occur at molar ratios.