Description

1.0 INTRODUCTION

Gabapentin primarily influences clinical and therapeutic outcomes in Human Astrocytes [1, 2]. However, there is a lack of research examining the growth and development of Human Astrocyte cells (HAC) when exposed to varying concentrations of gabapentin. Given the drug's increasingly significant role in treating a variety of clinical conditions, it presents a valuable subject for investigation [3, 4]. Additionally, the essential function of HACs in the growth, development, and operation of the central nervous system makes them an ideal focus for study. Therefore, exploring the interactions between gabapentin and HACs could reveal previously unknown insights regarding their behavior during cell growth and development. Since gabapentin is a pharmaceutical designed to target the central nervous system to produce therapeutic or potentially toxic effects, it is logical to further investigate its interactions with neural cells [2, 3, 4]. Consequently, a comprehensive experimental study on the impact of different molar concentrations of gabapentin on human astrocytes during their growth and post-maturation stages may clarify the structural, biochemical, and electro-molecular responses of HACs to such exposure. This is the primary aim of this research.

Gabapentin, originally developed for the treatment of epilepsy, has found applications in managing neuropathic pain, anxiety disorders, and other CNS-related conditions [2, 3, 4]. Its ability to modulate neurotransmitter release and influence neural excitability makes it a compelling subject for research, particularly in relation to astrocytes. Astrocytes are not merely supportive cells; they are integral to maintaining homeostasis, regulating blood flow, and modulating synaptic activity within the CNS [5]. Understanding how gabapentin affects these cells could unlock new therapeutic avenues and enhance our knowledge of its pharmacodynamics and potential toxicological effects.

Gabapentin has established itself as a cornerstone in the management of various neurological disorders, offering relief to countless patients suffering from conditions such as neuropathic pain, seizures, and postherpetic neuralgia. Its versatility extends beyond these primary indications, as it is frequently prescribed off-label for a range of other ailments, 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].

One of the remarkable features of gabapentin is its wide therapeutic dosage range, which can vary significantly from as low as 100 mg to as high as 3600 mg per day [3]. This flexibility allows healthcare providers to tailor treatment plans to individual patient needs, optimizing therapeutic outcomes. However, it is essential to note that some patients may develop tolerance to gabapentin over time. This phenomenon can necessitate dosage escalation to maintain the same level of efficacy, prompting careful monitoring and adjustment by healthcare professionals [6, 7, 8].

1.1 The Rising Tide of Gabapentin Prescriptions: A Double-Edged Sword

Between 2009 and 2016, the prevalence of gabapentin prescribing nearly doubled, with increases observed in every state across the United States, ranging from 44% to a staggering 179% [9]. By 2019, gabapentin had ascended to become the seventh most prescribed medication in the country, with a remarkable 69 million prescriptions issued that year alone [10]. While gabapentin is generally considered safe when used independently, its combination with opioids or benzodiazepines raises significant concerns, as it can potentiate the risk of overdose [9. 10]. This alarming trend highlights that the proliferation of gabapentin prescriptions is not without potential harm.

Recent studies, including a comprehensive analysis by the CDC, have shed light on the serious implications of gabapentin use. In a review of 62,652 overdose deaths that occurred between 2019 and 2020 across 24 jurisdictions, it was found that among the 58,362 deaths with documented toxicology results, gabapentin was detected in 5,687 cases (9.7%) [10]. Notably, gabapentin was involved in overdose deaths for 2,975 of these individuals, accounting for 52.3% of those with a positive gabapentin test result. The demographic characteristics of the decedents revealed a concerning trend: the majority of gabapentin-involved overdose deaths occurred among non-Hispanic White individuals (83.2%) and those aged 35 to 54 years (52.2%) [10].

These findings underscore a critical issue: more than half of the overdose deaths among individuals with a positive gabapentin toxicology test are directly related to the drug [10]. Despite its widespread use, the mechanisms underlying gabapentin toxicity, overdose, and mortality remain poorly understood [10]. This gap in knowledge calls for further research to elucidate the risks associated with gabapentin, particularly in combination with other central nervous system depressants.

As healthcare providers continue to navigate the complexities of gabapentin prescribing, it is essential to balance its therapeutic benefits against the potential for harm. Increased awareness and education regarding the risks of gabapentin, especially when used in conjunction with other medications, are crucial in mitigating the dangers associated with its use. The rising tide of gabapentin prescriptions serves as a reminder of the need for vigilance in prescribing practices and ongoing research to ensure patient safety in an evolving medical landscape.

Research scientists have proposed several multimodal theories to explain how this drug exerts its effects, particularly in relation to its structural similarities to amino acids. At the core of gabapentin's presumed functionality is its relationship with Gamma-Amino-Butyric-Acid (GABA) receptors especially those located on astrocytes. Although gabapentin is classified as a GABA analog, it is important to clarify that it does not directly bind to GABA receptors. Instead, its primary action involves the inhibition of voltage-gated calcium channels, specifically targeting the α2δ-1 subunit [1, 2, 4, 11]. This inhibition plays a crucial role in modulating neurotransmitter release, thereby influencing neuronal excitability and synaptic transmission.

Moreover, gabapentin's structural resemblance to branched-chain amino acids (BCAAs), particularly leucine, adds another layer of complexity to its mechanism. This similarity may facilitate interactions with various cellular pathways, further contributing to its therapeutic effects [1, 2, 4, 11]. Interestingly, gabapentin has also been shown to stimulate GABA synthesis through the action of glutamate dehydrogenase, enhancing the availability of this critical neurotransmitter [2, 4, 11]. Additionally, it may influence the transport of BCAAs, potentially impacting metabolic processes within the central nervous system.

Thus, while the exact mechanisms of gabapentin's function are still under investigation, its multifaceted interactions with calcium channels, GABA synthesis, and amino acid transport highlight its potential as a versatile therapeutic agent. Continued research is essential to unravel the intricacies of gabapentin's action, paving the way for more effective treatments for neurological disorders and a deeper understanding of its role within the complex landscape of neuropharmacology [2, 4, 11].

1.2 Why Human Astrocyte Cells (HACs)?

The exploration of Human Astrocyte Cells (HACs) is crucial for advancing our understanding of the central nervous system (CNS) and its intricate functions. Despite the growing body of research surrounding various pharmacological agents, the direct effects of gabapentin on HAC growth and development remain largely uncharted territory. Investigating this relationship is essential, as it may illuminate the broader implications of gabapentin on astrocyte behavior and function.

Astrocytes are the predominant cell type in the CNS, constituting a significant portion of the cellular volume—ranging from 25% to 50% in certain brain regions [5, 12, 13]. Remarkably, they outnumber neurons by more than fivefold [13], underscoring their importance in maintaining CNS homeostasis. These versatile cells exhibit diverse morphological and physiological forms, each tailored to fulfill specific roles in neural signaling and support. Recent studies have highlighted the involvement of astrocytes in synapse formation, synaptic function, and the regulation of blood flow within the brain, emphasizing their integral role in both normal and pathological processes [1, 2, 3, 12, 13].

While some research has indicated that gabapentin can inhibit the proliferation of colorectal cancer cells, particularly in HCT116 cell lines, similar investigations focusing on astrocytes during their growth and development are conspicuously absent. This gap in knowledge presents a unique opportunity to explore how gabapentin may influence HACs, potentially revealing novel insights into its therapeutic and toxicological effects [14].

Astrocytes are classified into two main subtypes: protoplasmic astrocytes, predominantly found in the gray matter, and fibrous astrocytes, which are primarily located in the white matter [15]. Both subtypes play vital roles in synaptic formation, neural cell development, and the regulation of cerebral blood flow. They are also key players in neural plasticity, a fundamental process that underlies learning and memory. Moreover, astrocytes express receptors for a variety of neurotransmitters and have the capacity to release numerous neuroactive and trophic factors, many of which remain to be discovered [16]. The functional significance of this signaling is still shrouded in mystery, but it is likely to be pivotal in controlling synaptic development, blood vessel dynamics, and neuronal survival [17, 18, 19, 20, 21, 22, 23, 24].

Thus, the study of Human Astrocyte Cells is not only essential for understanding the effects of gabapentin but also for unraveling the complex roles these cells play in the CNS. By investigating the interactions between gabapentin and HACs, we can gain valuable insights that may inform therapeutic strategies for a range of neurological diseases, ultimately enhancing our ability to treat and manage these conditions effectively [25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36].

1.3 Unraveling the Mysteries of Astrocyte-Secreted Signals and Neuronal Survival

Despite the wealth of literature surrounding astrocytes, many fundamental questions about the identities of the signals they secrete and their roles in promoting neuronal survival remain unanswered. Astrocytes may enhance neuron survival by facilitating the formation of synapses among central nervous system (CNS) neurons, or they might release specific signals that activate crucial neuronal survival pathways [16]. The complexity of astrocyte biology is underscored by transcriptomic analyses, which reveal a diverse array of trophic factors produced by these cells, suggesting their significant contribution to neuronal health. This intriguing area of research is ripe for exploration in future studies.

1.4 Revolutionizing Our Understanding of Gabapentin: A Focus on Cellular Mechanisms

As the scientific community delves deeper into the pharmacodynamics and mechanisms of action of gabapentin, a clearer picture is emerging of how this widely used medication influences cellular behavior. The molecular and biochemical changes triggered by gabapentin are pivotal in determining its effects on cell growth, viability, and overall survivability. These intricate pathways not only elucidate the drug's therapeutic potential but also highlight the risks associated with its use, including potential toxicological effects.

This study represents a significant shift in the trajectory of research within neurology and neuroscience. By moving away from broad, aimless investigations, we are now honing in on direct interventions at the cellular and tissue levels. This focused approach is not merely an academic exercise; it serves as a precursor to preclinical results that can expedite the often lengthy process of clinical studies.

Understanding the specific molecular pathways influenced by gabapentin allows research scientists to identify the precise mechanisms that underlie its effects. This knowledge is crucial for developing targeted therapies that maximize therapeutic benefits while minimizing adverse outcomes. By illuminating the connections between gabapentin's action and cellular responses, we can pave the way for more effective treatment strategies in managing neurological disorders.

Therefore, this research marks a transformative moment in our understanding of gabapentin. By prioritizing cellular and tissue-level interventions, we are setting the stage for innovative approaches that could significantly enhance patient care. As we continue to explore the complexities of gabapentin's pharmacological profile, we move closer to unlocking its full potential in the realm of neurology, ultimately improving outcomes for those who rely on this important medication.

2.0 METHODOLOGY

2.1 American Academy of Primary Care Research (AAPCR) Phase 1 Astrocyte Project: A Groundbreaking Study on Human Astrocyte Cell Culture

Experimental Design for Investigating the Effects of Gabapentin on Human Astrocyte Progenitor Cultures

In this study, human astrocyte progenitor lines were sourced from iXcell Biotech, with established protocols employed by the Southwest Research Institute (SwRI) to cultivate the cultures effectively [16, 17]. The experimental phase spanned a total of 11 days, during which meticulous monitoring of cellular developments was conducted to ensure accurate observations of growth and response to treatment. 

To facilitate a robust comparison, cultures were organized into duplicates, resulting in two control groups—Control A and Control B—and two gabapentin treatment groups—5 mM Gabapentin Group A and 5 mM Gabapentin Group B. All flasks were uniformly seeded with 5,000 cells/cm², totaling 375,000 cells per T-75 flask, and exhibited an impressive cell viability rate of 98.3% at the outset. Throughout the duration of the study, each flask was maintained at a constant volume of 10 mL of cell culture medium, tailored to the specific conditions of each group. To ensure the integrity of the experimental design, both the cell seeding concentrations and media volumes remained constant for the entirety of the study.

Daily media exchanges were performed to maintain optimal growth conditions, with the new media for the gabapentin groups consistently containing the predetermined concentration of 5 mM. This approach ensured that the concentration of gabapentin remained stable across all treatment flasks, allowing for a clear assessment of its effects on the growth and viability of human astrocyte progenitor cells.

 2.2 Preparation of Astrocyte Medium and Gabapentin Concentrations

The preparation of Astrocyte Medium was conducted following the protocol provided by iXcell. Initially, key components including Fetal Bovine Serum (FBS), Growth Supplement, and Antibiotic-Antimycotic (Anti-Anti) were thawed in a 37 °C water bath to ensure optimal conditions for mixing. To achieve a homogeneous mixture, the thawed components were gently tilted multiple times before incorporation into the medium. All subsequent steps were performed under aseptic conditions within a Biological Safety Cabinet (BSC) to maintain sterility. Specifically, 50 mL of FBS, 1 mL of Growth Supplement, and 5 mL of Antibiotic-Antimycotic were added to 500 mL of iXCell HA medium. The mixture was thoroughly combined to ensure even distribution of the components. The final concentrations in the Astrocyte Medium were established as follows: 10% FBS, 0.2% Astrocyte Supplement, and 1% Anti-Anti. Following the preparation of the Astrocyte Medium, drug-infused media formulations were created. To prevent contamination during cell culture, all media infused with non-sterile drugs were sterilized by filtration through a 0.22 μm sterile filter within the BSC [17, 18, 19, 20, 21, 22, 23, 24].

2.3 Gabapentin Media Solution Preparation

The gabapentin Media Solution was prepared using medical-grade gabapentin. For the creation of a 5 mM gabapentin solution, 300 mg of gabapentin was dissolved in 7.97 mL of the nutrient solution, resulting in a concentrated 220 mM solution. Subsequently, 4.55 mL of this 220 mM solution was diluted with 195.45 mL of the nutrient solution to achieve the desired final concentration of 5 mM. Each solution was then filtered through a 0.22 μm sterile filter to ensure sterility [18, 19].

3.0 RESULTS

3.1 Cell Indices: This comparative study of astrocyte cell growth examined two separate cell cultures: control A and control B, which were grown without the addition of gabapentin, and 5 mM gabapentin group A and group B, which included a 5 mM concentration of gabapentin in the culture media. All groups began with an equal cell count of 375,000, providing a consistent baseline for evaluating growth. Over the 11-day culture period, the data collected offered valuable insights into the proliferation patterns of human astrocytes in this experiment.

By the end of day 9, when the harvesting of control A and the 5 mM Gabapentin group A cells took place, the total cell count for control A had dramatically increased to an impressive 8.71 million (8,710,000) cells. In contrast,  the 5 mM gabapentin group A experienced only modest growth, reaching 3.22 million (3,220,000) cells.

By day 11, however, the analysis of control B cells showed a notable decline to 6.79 million (6,790,000) cells, reflecting a net loss of 1.92 million (1,920,000) cells, which corresponds to a 22% rate of cell apoptosis over the two-day period. Similarly, the 5 mM gabapentin group B showed a decrease to 2.72 million cells, indicating a net loss of 0.5 million (500,000) cells, representing a 15% rate of cell apoptosis within the same timeframe.

These findings underscore a significant change that occurred shortly after the initial similarities in growth patterns observed during the first nine days. Fig. 1,2,3 and 4

                                             Figure 1                                                                                                    Figure 2

                                         Figure 3                                                                                                          Figure 4

                                                                                     Control - blue dots;   5 mM Gabapentin  - orange dots

In this study, we meticulously collected and analyzed two primary sets of raw data to evaluate the effects of gabapentin on cell counts, cell size, viability and morphology. The first set of data comprises actual cell counts, size and viability measurements. These metrics provide a quantitative assessment of cell health and proliferation across both the control and gabapentin-treated groups. The results are summarized in Tables 1 to 7, which displays the cell indices for each group, allowing for a clear comparison of the effects of gabapentin treatment on HAC.

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

Table 2: Human Astrocyte cell viability on various days for the control and Gabapentin Groups

Table 3: Calculation of the Doubling Time (DT) from Study Raw Data using the formula

(DT) = T Ln2 / Ln (N9/N1)

Table 4: Calculation of the Mean Lifetime of Human Astrocyte Cells after the cells attained confluency.

3.2 Cell Electron Microscope Images:

The second set of data involves daily microscopy and photography conducted under standardized conditions across all T-Flasks. Utilizing a phase contrast electron microscope, we captured images at two magnifications (4X and 10X) to ensure comprehensive visualization of the cell cultures. These 4X images presented in Figures 5 to 8, provide an overview of the cell morphology and density, facilitating a broad assessment of the culture's health. The 10X images, while not displayed here, offer a more detailed view of individual cells, allowing for a closer examination of their structural integrity and any potential abnormalities.

The daily microscopy sessions served multiple purposes including morphological assessment, cell viability monitoring and culture contamination inspection, enabling direct visualization of cell shape and structure, direct observation of growth patterns and overall health of the cultures; and ensuring that no foreign life forms were present in the culture media, which could skew results [38]. The standardized microscopy records were crucial for making numerical comparisons using ImageJ software, a tool for assessment of cell density. This analysis not only enhances the reliability of our findings but also provides a robust framework for understanding the impact of gabapentin on cell culture behavior.

Thus, the combination of quantitative cell count, size and viability data, and the qualitative microscopy observations, formed a comprehensive dataset that underpins our investigation thereby highlighting the effects of gabapentin on HAC cultures.

The use of phase-contrast electron microscopy to study cellular growth patterns has proven to be an essential method, providing remarkable insights into the dynamics of experimental cultures. The images captured (Figures 5 to 8) displayed strikingly similar growth patterns for both control A and control B, as well as for gabapentin group A and group B, facilitating a valuable side-by-side comparison across the culture flasks. This careful methodology not only reinforced our findings but also ensured internal and external consistency [34, 35, 36, 38].

Figures 5, 6, 7, and 8 are Displayed Below: Showing real time Comparative Phase Contrast Electron Micrograph Images of HAC for Control Groups and 5mM Gabapentin groups as Labeled

Figure 5

Figure 6

Figure 7

Figure 8

Any anomalies detected within these cultures should trigger a comprehensive internal review of the procedural methods and the various environmental factors that may influence cell proliferation. In this context, any irregularities or unexpected changes in flask conditions necessitate a thorough audit to maintain the reliability of the results. No such anomalies were observed during this experiment, indicating a high level of methodological precision.

Notably, the morphological details observed confirmed that control A and control B maintained identical structures from day 1 to day 9, as shown in Figure 8. Similarly, the morphological details observed for gabapentin group A and group B displayed the same growth pattern during this period. However, a significant change occurred when we examined control B and gabapentin group B on days 10 and 11, just two days after confluence. These images revealed a remarkable transformation compared to the cells at confluence (day 9), marking a crucial moment that could provide insights into growth regulation (Figures 5 to 8). This transformation opens the door to new hypotheses regarding downstream effects on cellular behavior, paving the way for further research in future studies [37].

In the early snapshots of cellular development, vivid imagery showcases the fascinating structure of astrocytes. By day 2, these cells displayed a striking star-like shape, with long, delicate connecting fibers extending outward like the arms of celestial bodies in a cosmic dance. Each branch intertwines to form networks vital for communication and support, highlighting the intricate design behind the seemingly ordinary yet extraordinary world of neurons.

As we progress to days 4 and 5, the narrative took a vibrant turn—astrocytes began to take on a granule-like appearance. Within these cells, a surge of activity suggests an underlying physiological shift. These granules remain a biological mystery, yet they may signify a period of heightened metabolism, preparing the astrocytes for subsequent exponential growth. Like seeds ready to sprout, these granules could indicate an impending flourish, a precursor to a complex symphony of cellular and molecular synthesis. As we journey through cellular evolution, we cannot help but marvel at the parallels of life—the microcosmic struggles and triumphs echoing those in the vast universe beyond. Whether in stillness or dynamic change, the story of these astrocytes resonates deeply, reminding us of the relentless energy propelling life forward, both within and around them.

On days 7 and 8, our exploration unveiled a rich tapestry of cellular activity within the culture flasks, characterized by lush cell cytoplasm and clear signs of growth and maturation. The cells exhibited significant granulation and interconnections, indicating a transformative phase in their development. By day 9, the human astrocytes displayed an intriguing abundance of fibrillary content, their appearance marked by delicate, whitish appendages—often linear—that created a captivating silhouette against the vibrant intercellular landscape. These linear extensions appear to signal a critical aspect of astrocyte matrix organization, reflecting their evolving complexity and maturity. The morphology observed allows neuro scientists to gain a deeper understanding of astrocytic roles in neural environments. This research engaged in a nuanced discussion about the precise timing of confluence; while the vibrant images and slides suggest an ongoing transition, pre-culture investigations strongly indicate day 9 as the definitive confluence point. Herein lies the intersection of observation and interpretation, weaving together the narrative of cellular evolution as we stand on the brink of understanding the rich tapestry of neuroglial biology. Each day reveals a new story, underscoring the dynamic interplay of maturation, complexity, and organization that defines the life of astrocytes in culture.

In reviewing the series of microscopic images captured over eleven days, a remarkable transformation in the cellular landscape becomes apparent—especially on day 10. The emergence of numerous vesicles sharply contrasts with their scarce presence in the earlier images from days 1 through 9. These vesicles, typically associated with cellular communication and transport processes, indicate a shift in the state of the observed cells. Additionally, the image from day 10 shows notable turbidity and subtle discoloration in the slides, characteristics often linked to the aging process. This change may suggest a decline in cellular integrity or an accumulation of metabolic byproducts, both serving as strong indicators of diminishing cellular health.

The insights take a more dramatic turn on day 11. Here, the microscopic view reveals further advancements in these traits: vacuoles appear alongside conspicuous empty spaces within the culture area, resembling conditions similar to astrogliosis observed in stressed brain tissues [39, 40, 41, 42]. The presence of vacuoles typically indicates intracellular changes as the cells respond to injury or metabolic disturbance, a marker of cellular toxicity [43]. These findings warrant further investigation and highlight the importance of real-time monitoring in understanding the evolution of cellular architecture over time, particularly concerning neurodegenerative diseases or age-related declines. The images serve as a compelling visual narrative, chronicling the intricate and often unraveling stories etched within each cell as they navigate the challenges of aging.

4.0 DISCUSSION 

4.1 Reliability and Reproducibility

The multiple layers of controls and pairs in this experimental design was used to enhance reproducibility and reliability, reduce inter-observer variability, and produce valid results based on systematic application of intervention while keeping other variables constant. Experiments should not be designed only to satisfy a statistical exercise by creating triplicates to demonstrate statistical measures of centrality like means, standard deviations and more for a group of data, and then fail to answer any scientific question for which it was designed to do. This raises questions on the use of triplicates in cell culture protocols [37]. When the intraclass correlation coefficient (cf) is greater than 75% (0.75) like in this study, there is no gain to make triplicate observations [34]. The correlation coefficient in this study was > 0.95 discouraging the use of triplicate. 

Similarly, when cell cultures are grown from same or similar cell lines and the growth protocols are congruent and identical, in such cases it is well expected that a large correlation coefficient is certain especially when the culture conditions are identical. In our study it is well established that HAC from iXcells have already known growth pattern under the same culture media provided and applying the same culture protocol as prescribed [37].

This approach for conducting experiments on cell lines by interlinking cell culture and dose-response study experiments like we have done with gabapentin here is certainly unique and innovative. It allows us to design the experiment embedding, at the same time, intra and inter experimental checks and controls that validate reliability and reproducibility of the experiment. [35, 36] This experimental design is simple and cost effective limiting the number of technical and biological replicates needed to show reproducibility by building internal and external controls and by incorporating an escalating dose response model as a testament to internal validity and reproducibility [37].

4.2 The Effects of Gabapentin on HAC Growth

This study examined the effects of gabapentin on human astrocyte cells (HAC) and significant findings emerged regarding the drug's influence on cell growth and viability. When compared to control groups, gabapentin was shown to decrease the exponential growth rate of HAC prior to reaching confluence (fig. 3 and 4); confluency is a critical phase in cell culture when cell growth covers the entire surface area of the culture vessel. Overall gabapentin suppressed the growth of HACs compared to controls; at a concentration of 5mM, gabapentin caused a marked reduction in the growth rate when compared to the control group.

In addition, after confluence phase on day 9, both control group B and the 5 mM gabapentin group B experienced a notable attrition of cells. By day 11, the control B group saw a decline in cell numbers from an initial 8.72 million at confluence to 6.79 million, reflecting an increased rate of apoptosis among astrocyte cells. Specifically, between days 9 and 11, approximately 22% of the astrocytes in the control group underwent apoptosis. Similarly, the 5mM gabapentin group B exhibited a significant decrease in HAC numbers, dropping from 3.22 million on day 9 to 2.72 million by day 11, which represents a 15% decline in cell count. These findings underscore the inhibition by gabapentin of human astrocyte cell growth, highlighting its potential role in influencing cellular dynamics in neurobiological research.

This study provides compelling evidence that gabapentin at a concentration of 5mM not only reduces the growth rate of human astrocyte cells but also increases the rate of apoptosis, leading to a significant decline in cell numbers at confluency and post-confluency. Further research may explore the underlying mechanisms of these effects and their implications for therapeutic applications.

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, two distinct groups were established: a control group cultured without any pharmaceutical intervention and a gabapentin group treated with 5 mM gabapentin. By maintaining all other conditions constant, the study aimed to isolate the effects of gabapentin on astrocyte growth pattern.

From as early as day 2, observable differences in cell growth and density were noted in the astrocyte cultures, as illustrated in the T-75 flask images (Figures 5 to 8). The 5 mM gabapentin groups exhibited a slightly lower cell density compared to the control groups, a trend that persisted throughout the culture period and that became more pronounced over time. This oligemic appearance in the gabapentin groups suggested that the 5 mM concentration of gabapentin effectively reduced the growth rate of astrocytes. However, it is noteworthy that the growth pattern remained sigmoidal, with both groups reaching confluency by day 9. This critical observation indicates that gabapentin did not influence the time to confluency in astrocyte cultures, challenging the dogmatic notion in cell biology that cell culture confluency is determined by the available surface area.

Traditionally, it has been posited in cellular biology that adherent cells, such as human astrocytes, cease growth upon contact with themselves and with limiting culture surface area, this is the phenomenon of contact inhibition [44, 45]. The fact that the gabapentin group reached confluence simultaneously with the control group calls into question this long-held dogma, suggesting that external agents like gabapentin can modulate cell counts at confluence and beyond without affecting the time to reach confluence. From mathematical logic, however, one would expect that when cell growth rate slows down and precocious aging sets in then the time for the culture to cover the plate’s surface area would be prolonged, but this was not the case. 

4.4 The Effect of Gabapentin on Cell Size

In cellular biology, cell size is often regarded as a marker of aging, with cells typically increasing in size as they age [37, 46]. In this study, a similar trend was observed. The control group astrocyte cell size increased from 14.8 µm on day 1 to 15.7 µm by day 9, reflecting a modest 6% increase. By day 11, the size further increased to 15.8 µm, marking a 6.7% increase overall. In contrast, the 5 mM gabapentin group demonstrated a more pronounced increase in cell size, reaching 16.6 µm by day 9—a 12.2% increase—and 17.0 µm by day 11, representing a 14.9% increase. The difference in cell size between the two groups, the control and 5mM gabapentin groups, was statistically significant (p = 0.033), suggesting that gabapentin may accelerate the aging process of astrocyte cells. While the drug appears to reduce growth rates and increase cell size and cause cells to age, ironically it does not alter the time to achieve confluence as discussed above.

4.5 The Effect Gabapentin on HAC Culture Longevity

Shifting focus to the post-confluence phase, the decline in cell growth was quantified using the decay constant equation (Table 4). The control group exhibited a decay constant rate (λ) of 4,590 cells/hr, while the gabapentin group showed a lower decay constant rate of 3,230 cells/hr. This translates to a mean lifetime of 218 hours for the control group astrocytes, compared to 309 hours for the gabapentin group—an increase of 91 hours in survival time that was statistically significant (p = 0.024). This finding indicates that astrocyte cells exposed to 5 mM gabapentin not only survived longer but also exhibited a notable difference in longevity compared to their untreated counterparts.

Our findings suggest that gabapentin at a concentration of 5 mM may have significant implications on the mean lifetime expectancy of human astrocyte cells (HACs). This concentration could either enhance the resilience of these cells, allowing them to adapt to metabolic stress, or it may induce a level of stress that leads to 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 also be a critical biological process in the cells.

Furthermore, the increased longevity of astrocytes in the presence of gabapentin raises important questions about the drug's role in cellular aging and its potential implications for therapeutic applications. 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

One of the critical insights from this study is that the administration of gabapentin in molar concentrations was essential for achieving consistent and predictable biological responses in this study, a not so surprising phenomenon since molecular interactions in chemistry occur in molar ratios. The results clearly indicate that gabapentin at 5 mM significantly suppressed the growth of HACs, and this effect is dependent on molar concentrations. The predictable responses observed in this study highlight the necessity of measuring gabapentin concentrations in molar ratios like millimoles (mM), and without this most critical design component, the cellular and chemical interactions will not demonstrate the same clarity, underscoring the importance of molar ratios in both chemical and biological systems. This also suggests that in biological systems cell to cell interactions also occur in molar ratios [47].

The clinical implications of these findings are profound. Currently, gabapentin is prescribed in as weight based measures such as milligrams and grams, which may not produce the consistent and predictable responses seen at the cellular level like in this study. Understanding that chemical and molecular reactions occur in molar ratios is crucial, especially in pharmacology and clinical medicine. This knowledge could lead to more effective dosing strategies that align with the observed cellular responses.

The suppression of HAC growth observed with increasing doses of gabapentin reflects the drug's toxicity pattern towards human astrocyte cells. It is important to note that the gabapentin used in this research is identical to that prescribed by physicians, highlighting the need for a deeper exploration of its toxicology in vivo, particularly at millimolar 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, a thorough understanding of its therapeutic and toxicological profiles is essential to ensure patient safety and maximize therapeutic benefits.

These results indicate that gabapentin, at 5 mM concentration, can lead to varying levels of HAC growth suppression and promote apoptosis. This raises critical questions about the mechanisms underlying gabapentin's effects. Does this mechanism contribute to its efficacy in suppressing neuropathic pain? Furthermore, could it play a role in the potentiation of addiction when gabapentin is combined with other addictive medications? Addressing these questions through focused research could significantly enhance our understanding of the pharmacology of gabapentin, the therapeutic applications, and the 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

This study has demonstrated that human astrocyte cell (HAC) cultures treated with gabapentin at 5mM concentration offer significant insights into the drug's effects on cell growth and development. The findings highlight the potential for gabapentin to be utilized in innovative ways within clinical pharmacology, therapeutics, and toxicology, ultimately enhancing its application in clinical medicine. The results indicate that while gabapentin reduces growth rates and increases cell size, it does not affect the time to confluency in human astrocyte cultures. This observation challenges existing assumptions about the relationship between cell density and growth dynamics, poking a hole on the dogma that final cell count of a biological culture is determined by contact inhibition with the culture plate surface area. Moreover, the increased longevity of astrocytes exposed to gabapentin prompts critical inquiries into the drug's role in cellular aging and its broader therapeutic implications. As we look to the future, it is essential for subsequent research to explore the underlying mechanisms of these findings. A deeper understanding of gabapentin's multifaceted effects on cellular biology could pave the way for more effective treatment strategies. Additionally, this study advocates for the use of drugs in molar concentrations ratios to achieve more predictable clinical and toxicological responses. By embracing this approach, we can enhance the safety and efficacy of pharmacological interventions, ultimately benefiting patient care and outcomes.